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K G Mann

Publications and source records attributed to K G Mann.

At least 91 records · Page 5Linked to original sources
PubMed1996

Insights into the complex association of bovine factor Va with acidic-lipid-containing synthetic membranes.

The mechanism of binding of blood coagulation cofactor factor Va to acidic-lipid-containing membranes has been addressed. Binding isotherms were generated at room temperature using the change in fluorescence anisotropy of pyrene-labeled bovine factor Va to detect binding to sonicated membrane vesicles containing either bovine brain phosphatidylserine (PS) or 1,2-dioleoyl-3-sn-phosphatidylglycerol (DOPG) in combination with 1-palmitoyl-2-oleoyl-3-sn-phosphatidylcholine (POPC). The composition of the membranes was varied from 0 to 40 mol% for PS/POPC and from 0 to 65 mol % for DOPG/POPC membranes. Fitting the data to a classical Langmuir adsorption model yielded estimates of the dissociation constant (Kd) and the stoichiometry of binding. The values of Kd defined in this way displayed a maximum at low acidic lipid content but were nearly constant at intermediate to high fractions of acidic lipid. Fitting the binding isotherms to a two-process binding model (nonspecific adsorption in addition to binding of acidic lipids to sites on the protein) suggested a significant acidic-lipid-independent binding affinity in addition to occupancy of three protein sites that bind PS in preference to DOPG. Both analyses indicated that interaction of factor Va with an acidic-lipid-containing membrane is much more complex than those of factor Xa or prothrombin. Furthermore, a change in the conformation of bound pyrene-labeled factor Va with surface concentration of acidic lipid was implied by variation of both the saturating fluorescence anisotropy and the binding parameters with the acidic lipid content of the membrane. Finally, the results cannot support the contention that binding occurs through nonspecific adsorption to a patch or domain of acidic lipids in the membrane. Factor Va is suggested to associate with membranes by a complex process that includes both acidic-lipid-specific and acidic-lipid-independent sites and a protein structure change induced by occupancy of acidic-lipid-specific sites on the factor Va molecule.

Adsorption↗
PubMed1996

Regulation and regulatory role of gamma-carboxyglutamic acid containing clotting factors.

Blood coagulation is initiated following damage of the vessel wall. The large number of proteins that participate in the reactions that regulate blood coagulation must behave in a concerted manner to generate alpha-thrombin. This most abundant enzyme product of the coagulation process acts on the soluble blood protein fibrinogen to create the insoluble fibrin clot, trigger platelet activation, and initiate a variety of vascular processes ranging from coagulation inhibition and activation of fibrinolysis to cell growth. The majority of enzymes that participate in the blood-clotting process circulate in blood as inactive zymogens and procofactors. Following mechanical injury to the vessel wall, a cell-associated protein, tissue factor, is exposed and initiates a series of enzymatic reactions localized on a membrane surface generally provided by cells and accumulating platelets. The binding of the enzymes and zymogen substrates to the exposed membrane surface and their activation are in part governed by the gamma-carboxyglutamic acid residues (gla) of each protein. Recently, it has been demonstrated that the gla residues are necessary for Ca2+ binding and subsequent exposure of the hydrophobic membrane binding regions to the cell bilayer. Thus, the gla residues play a preeminent role during the blood-clotting process. Absence of gamma-carboxylation of one or more of the critical glutamic acid residues results in an impaired coagulation/anticoagulation process which may lead to a bleeding diathesis or thrombosis.

1-Carboxyglutamic Acid↗
PubMed1995

Factor V turnover in a primate model.

The isolation and characterization of baboon plasma factor V (FV) were performed for the development of an in vivo model for studying factor V/Va physiology in nonhuman primates. Baboon FV was purified by immunoaffinity chromatography with an antihuman FV monoclonal antibody and exhibits a specific activity of 1,940 U/mg. Baboon FV activation by thrombin proceeds through two proteolytic pathways similar to those observed with human and bovine FV. Limited amino acid sequencing of FV and its thrombin activation fragments shows 95% identity with human and 79% identity with bovine FV. 125I-Factor V and a mixture of thrombin cleaved 125I-FV activation products were infused into normal male baboons and evaluated by blood sample radioactivity measurements and by autoradiography of plasma samples following resolution by gel electrophoresis. Factor V disappeared with a half-life (t1/2) of 12.98 +/- 1.85 hours and was cleared without obvious degradation of the molecule during circulation. The radioactivity associated with the thrombin activated FV mixture, which consisted of the Mr = 220,000 activation intermediate, the Mr = 150,000 activation peptide, the heavy chain (HC) and the light chain (LC) of FVa, was cleared in a nonlinear manner. The HC and LC were removed with t1/2 < 20 minutes. The apparent molecular weight (Mr) = 220,000 and Mr = 150,000 fragments were cleared with t1/2 > 6 hours and t1/2 > 30 hours, respectively.

Animals↗
PubMed1995

The mechanism of inactivation of human platelet factor Va from normal and activated protein C-resistant individuals.

The inactivation of human platelet factor Va by activated protein C (APC) was analyzed by functional assessment of cofactor activity and Western blotting analysis to visualize the factor Va fragments accompanying proteolysis. Platelets were treated with thrombin to facilitate both their activation as well as the release and further activation of platelet factor Va, followed by APC addition. The rates of inactivation were donor-dependent such that 15-60% of the initial cofactor activity was lost within 5 min of APC addition with as much as 10-20% of the activity still remaining after 2 h of incubation. Western blot analysis using a monoclonal antibody that recognizes an epitope between amino acid residues 307 and 506 of the factor V molecule suggested that the factor Va activity resistant to APC inactivation was due to residual heavy chain. Furthermore, in contrast to studies with normal plasma-derived factor Va, two possible cleavage mechanisms could explain the platelet factor Va fragments observed. APC can cleave platelet factor Va initially at Arg506, with subsequent cleavages occurring at Arg306 and Arg679. Alternatively, APC can cleave at Arg306 initially, with further cleavage at Arg679 then at Arg506 or at Arg506 followed by cleavage at Arg679. Similar results were obtained if platelets were removed from the inactivation mixtures and phospholipid vesicles were used to supply the membrane surface required for inactivation, suggesting that the order of platelet factor Va peptide bond cleavage or the amount of cofactor activity remaining was not altered by either of these surfaces. Thus, APC is unable to effect the complete inactivation of platelet factor Va, even though it would appear that the same cleavages which render the plasma cofactor inactive are occurring in the platelet cofactor. Analogous protocols were used to study an individual heterozygous for the Arg506-->Gln506 mutation (Factor V Leiden, Factor VR506Q). With respect to the mutant platelet factor Va in the presence of APC, > 70% of the initial cofactor activity remained after 1 min, with 30% activity still remaining after 2 h. As seen in studies of the APC-catalyzed inactivation of plasma factor VaR506Q, proteolysis of the mutant platelet factor Va confirms that even though cleavage at Arg306 will occur in the absence of cleavage at Arg506, the rate of inactivation is slower. Collectively these data suggest that when compared to normal plasma factor Va, differences in normal platelet factor Va which define: 1) whether the heavy chain is susceptible to cleavage at Arg306 or Arg506 and 2) the extent to which it is cleaved initially at Arg306, in contrast to cleavage of Arg506, will define both the extent and rate of inactivation.

Amino Acid Sequence↗
PubMed1995

Factor VNew Brunswick: Ala221-to-Val substitution results in reduced cofactor activity.

We have characterized the factor V protein and cDNA of a patient displaying factor V deficiency (parahemophilia) and correlated the reduced activity with a missense mutation of Ala221-to-Val. Plasma from the subject individual (C1) presented reduced factor V antigen (39% of normal) that displayed reduced activity (approximately 26% of normal). Factor V purified from this individual by standard techniques shows normal migration on sodium dodecyl sulfate gels and a normal pattern of activation by thrombin. Purified antigen from sibling C2 gives a much reduced specific activity of 263 U/mg (17% of normal). Sibling C3, the mother, and the father have antigen within the normal range (57% to 200%) that has approximately normal specific activity. The cDNA encoding the factor Va heavy and light chains of the subject individual was polymerase chain reaction-amplified and sequenced and revealed an A-to-G substitution at position 3 of codon 51 (silent mutation), a C-to-T substitution in position 2 of codon 221 (Ala221-Val), a T-to-C substitution at position 3 of codon 708 (silent mutation), and a G-to-A substitution at position 1 of codon 2185 (Thr2185-Ala). The latter mutation was also observed in control individuals and is proposed to be a possible polymorphism. Restriction analyses demonstrated the presence of one mutant and one normal allele in the father. The subject individual (C1) and sibling C2 carry only the mutant allele. The mother and sibling C3 carry only the normal allele. The inheritance pattern suggests the presence of a missing or nonexpressed allele in the mother that is passed on to all the siblings. Expression of only the mutant allele by the subject individual (C1) and sibling C2 is consistent with reduced factor V antigen and activity in these patients. We have designated this mutant as Factor VNew Brunswick.

Adolescent↗
PubMed1995

Fluorogenic substrates for activated protein C: substrate structure-efficiency correlation.

A series of 87 fluorescent peptide substrates have been synthesized and evaluated using human and bovine activated protein C (APC). These substrates contain various isomers of aminonaphthalenesulfonamides (ANSN) as the detecting group and were substituted at P4, P3, P2, P'1, and P'2 positions. Substrates with 6,2-ANSN at P'1 had the highest fluorescence quantum yield, exceeding that of 6,1-ANSN 9.3-fold and 5,1-ANSN almost 1000-fold. Almost 50 substrates containing substituted ANSNs as leaving groups have KM values for APC below 100 microM, reaching as low as 4-10 microM for 8 of these substrates. These values are significantly lower than those reported for p-nitroanilide and 4-methylcoumaryl-7-amide substrates. Additionally, some of these substrates have relatively high kcat exceeding 50 s-1. These constants as well as kcat/KM are influenced by the nature of amino acid in the P3 and P2 positions, by the isomer of ANSN (P'1), and by the structure of substituent incorporated in the sulfonamide moiety (P'2). The highest kcat/KM were found for substrates with D-isomers of Leu, Phe, and Val in the P3 position when these amino acids were N-unblocked. For the P2 position Val, Phe, and Leu were preferable. Substrates containing n-butyl (bovine APC) and benzyl (human APC) substituents in the (P'2) structure have elevated kcat/KM. ANSN-containing substrates are hydrolzyed by both human and bovine APC at a similar rate.

Amino Acid Sequence↗
PubMed1995

Characterization of the molecular defect in factor VR506Q.

A poor anticoagulant response of plasma to activated protein C is correlated with a single mutation in the factor V molecule (Arg506-->Gln). Factor V was purified to homogeneity from plasma of two unrelated patients (patient I, factor VI, and patient II, factor VII), who are homozygous for this mutation. The factor V molecule from both patients has normal procoagulant activity when compared with factor V isolated from normal plasma in both a clotting time-based assay and in an assay measuring alpha-thrombin formation. The cleavage and subsequent inactivation by activated protein C (APC) of the alpha-thrombin-activated membrane-bound cofactor (factor Va) from both patients were analyzed and compared with the cleavage and inactivation of normal human factor Va. In normal factor Va, cleavage at Arg506 generates a M(r) = 75,000 fragment and a M(r) = 28,000/26,000 doublet and is necessary for the optimum exposure of the sites for subsequent cleavage at Arg306 and Arg679. Proteolysis at these sites leads to the appearance of M(r) - 45,000 and 30,000 fragments and a M(r) = 22,000/20,000 doublet. Cleavage at Arg306 is membrane-dependent and is required for complete inactivation. Following 5 min of incubation with APC (5.4 nM) membrane-bound normal factor Va (280 nM) has virtually no cofactor activity whereas under similar experimental conditions factor VaI and factor VaII retain approximately 50% of their initial activity. After 1 h of incubation with APC, factor VaI retains 20% of its initial cofactor activity whereas factor VaII has 10% remaining cofactor activity. The initial loss in cofactor activity (approximately 70%) of membrane-bound factor VaI and factor VaII during the first 10 min of the inactivation reaction is correlated with cleavage at Arg306 and appearance of a M(r) = 45,000 fragment and a M(r) = 62,000/60,000 doublet. Subsequently, the M(r) = 62,000/60,000 doublet is cleaved at Arg679 to generate a M(r) = 56,000/54,000 doublet resulting in complete loss of cofactor activity. Both procofactors, factor VI and factor VII, were inactivated following cleavage at Arg306 and Arg679, with APC inactivation rates equivalent to those observed for normal factor V. Our data demonstrate that: 1) cleavage at Arg506 is required for optimum exposure of the cleavage sites at Arg306 and Arg679 and rapid inactivation of membrane-bound factor Va; and 2) cleavage at Arg306 by APC on membrane-bound factor V occurs at the same rate in both normal and APC-resistant individuals. Thus cleavage at Arg306 and Arg679 and subsequent inactivation of the membrane-bound procofactor, factor V, does not require prior cleavage at Arg506 for optimum exposure.

Arginine↗
PubMed1995

Biochemical prototype for familial thrombosis. A study combining a functional protein C mutation and factor V Leiden.

Resistance to activated protein C (APC) is associated with a single amino acid substitution in factor V (Arg506-->Gln, factor V Leiden) that results in delayed inactivation of the molecule by APC. The mutation is present in 20% of patients with a first episode of deep venous thrombosis. Arterial and venous thromboses are also associated with the type II protein C deficiency (protein CVermont). In protein CVermont, the substitution Glu20-->Ala alone (rPC gamma 20A) is responsible for the defective anticoagulant properties of PCVermont. It was recently established that a thrombotic episode occurred in 73% of family members who are heterozygous for both a functional protein C gene mutation and the factor V Leiden mutation. We evaluated the molecular defect that would accrue in the combined deficiency state of factor VR506Q/VaR506Q and rAPC gamma 20A using recombinant APC and natural purified factor VR506Q from patients homozygous for the Arg506-->Gln substitution. While wild-type recombinant APC (rAPC) slowly cleaves and inactivates factor VR506Q and factor VaR506Q, minimal cleavage of membrane-bound factor VR506Q and VaR506Q by rAPC gamma 20A at Arg306 and Arg679 occurs, and no loss in cofactor activity is observed. Our data demonstrate that rAPC gamma 20A cannot inactivate either factor VR506Q or factor VaR506Q at biologically relevant rates because of impaired cleavage at Arg306 and Arg679.(ABSTRACT TRUNCATED AT 250 WORDS)

Factor V↗
PubMed1995

Evidence that meizothrombin is an intermediate product in the clotting of whole blood.

Meizothrombin is an intermediate that is produced during the conversion of prothrombin to thrombin in systems composed of purified factor Xa and factor Va that are quantitatively assembled on an anionic phospholipid surface. The biological significance of this intermediate has recently been challenged by the apparent absence of meizothrombin during clotting of sodium citrate-anticoagulated plasma. We analyzed the formation of thrombin during coagulation of nonanticoagulated, unchilled, minimally manipulated whole blood in glass tubes. The process was stopped at 0, 3, 5, and 7 minutes by the addition of biotinylated peptidyl chloromethyl-ketone active-site labeling reagents. Plasma/serum was separated by centrifugation, and labeled species were extracted by immunoadsorption with a polyclonal anti-prothrombin antibody. The purified prothrombin-derived species were separated by SDS-polyacrylamide gradient gel electrophoresis and visualized on a chemiluminescent avidin blot. Meizothrombin appeared as an intermediate product of this reaction and persisted with some increase through the 7-minute time point. We also observed incorporation of the active-site label into a species of lower molecular weight consistent with the B1 chain of beta- and/or gamma-thrombin. These degraded forms of thrombin have not been previously demonstrated in a biologically relevant preparation. Our data clearly establish the generation of meizothrombin as an intermediate product of thrombin generation during whole-blood clotting. The data also represent the first experimental evidence for the generation of beta- and gamma-thrombin in a biologically relevant environment and time scale.

Amino Acid Chloromethyl Ketones↗
PubMed1995

Alpha-thrombin stimulates urokinase production and DNA synthesis in cultured human cerebral microvascular endothelial cells.

alpha-Thrombin regulation of endothelial cell (EC) fibrinolysis has been documented by using endothelia derived from a number of anatomic locations but not with those derived from the human cerebral vasculature. In the present study, the fibrinolytic properties of human cerebral microvascular ECs and their regulation by alpha-thrombin are delineated and contrasted with those of human umbilical vein and foreskin microvascular ECs. In cerebral ECs, alpha-thrombin elicited a unique dose-dependent increase in urokinase production and DNA synthesis. Maximal stimulation, observed with 10 nmol/L alpha-thrombin, resulted in a 30- to 50-fold increase in urokinase production and a concomitant fourfold increase in DNA synthesis; the increase in urokinase was reflected in higher steady-state levels of urokinase mRNA. The major urokinase product secreted is the single-chain form of the enzyme. No effect was observed with the addition of other proteases or catalytically inactive variants of alpha-thrombin. A thrombin receptor agonist peptide upregulated urokinase production but had no effect on DNA synthesis, suggesting that fibrinolysis is mediated by the thrombin receptor but that proliferation is regulated by a different pathway. These findings suggest the possibility that the cerebral microvasculature may be a specialized region of the vascular system in which urokinase-type plasminogen activator, not tissue-type plasminogen activator, is the key catalyst of fibrin lysis when the brain responds to thrombotic events and that alpha-thrombin may regulate repair of the cerebral microvascular system.

Blotting, Northern↗
PubMed1995

Regulation of human bone marrow-derived osteoprogenitor cells by osteogenic growth factors.

Human bone marrow contains a distinct cell population that expresses bone proteins and responds to transforming growth factor beta 1 (TGF-beta), but not to hematopoietic growth factors (Long, M. W., J. L. Williams, and K. G. Mann. 1990. J. Clin. Invest. 86:1387-1395). We now report the isolation, characterization, and growth factor responsiveness of these precursors to human osteoblasts and the identification of a human osteoprogenitor cell. Immunological separation of human bone marrow nonadherent low-density (NALD) cells results in a marked enrichment of cells that express osteocalcin, osteonectin, and bone alkaline phosphatase. Flow cytometric analyses show that distinct cell subpopulations exist among these isolated cells. The majority of the bone antigen-positive cells are approximately the size of a lymphocyte, whereas other, less frequent antibody-separated subpopulations consist of osteoblast-like cells and osteoprogenitor cells. In serum-free cultures, TGF-beta stimulates the small, antigen-positive cells to become osteoblast-like, as these cells both increase in size, and express increased levels of osteocalcin and alkaline phosphatase. Antibody-separated cells also contain a separate population of clonal progenitor cells that form colonies of osteoblast-like cells when cultured in serum-free, semi-solid media. Two types of human osteoprogenitor cells are observed: a colony-forming cell (CFC) that generates several hundred bone antigen-positive cells, and a more mature cluster-forming cell that has a lesser proliferative potential and thus generates clusters of 20-50 antigen-positive cells. Osteopoietic colony-forming cells and cluster-forming cells have an obligate but differential requirement for osteogenic growth factors. The CFCs respond to TGF-beta, basic fibroblast growth factor (bFGF), bone morphogenic protein-2 (BMP-2), and 1, 25-dihydroxy vitamin D3 (1,25-OH D3). In contrast to the colony-forming cells, cluster-forming cells are regulated predominantly by 1,25-OH D3 and TGF-beta, but fail to respond to bFGF. We conclude that human bone marrow contains a nonhematogenous, heterogeneous population of bone precursor cells among which exists a population of proliferating osteoprogenitor cells. Further characterization of these bone precursor cell populations should yield important information on their role in osteogenesis in both health and disease.

Alkaline Phosphatase↗
PubMed1994

The mechanism of inactivation of human factor V and human factor Va by activated protein C.

The cleavage of human factor V and human factor Va by human activated protein C (APC) was analyzed in the absence and presence of phospholipid vesicles containing 75% phosphatidylcholine (PC) and 25% phosphatidylserine (PS). Membrane-bound human factor V (250 nM) is cleaved by APC (2.5 nM) to give M(r) = 200,000, 70,000, 45,000, and 30,000 fragments and an M(r) = 22/20,000 doublet. These fragments are released after four sequential cleavages of the membrane-bound procofactor at Arg306, Arg506, Arg679, and Lys994. No cofactor activity is observed following thrombin treatment of the membrane-bound APC-cleaved procofactor. In the absence of a membrane surface, no cleavage of factor V by APC is observed, and following thrombin activation factor Va retains full cofactor activity. Membrane-bound human factor Va (600 nM) loses more than 90% of its initial cofactor activity after 10 min of incubation with APC (10.9 nM), and virtually no cofactor activity is observed after 1 h of incubation. Under similar conditions but in the absence of PCPS vesicles, factor Va is cleaved but retains approximately 80% of its initial cofactor activity after 2 h of incubation with APC. In the presence of PCPS vesicles, the APC related loss of activity is correlated with cleavage of the heavy chain and appearance of fragments of M(r) = 45,000, 30,000, and of 28/26,000, and 22/20,000 doublets. These products correspond to three cleavages of the heavy chain (at Arg306, Arg506, and Arg679). Cleavage at Arg506 of factor Va precedes and appears to be required for cleavage at Arg306 and Arg679. In the absence of membrane, proteolysis at Arg506 produces an M(r) = 75,000 fragment which corresponds to the NH2-terminal portion of the human factor Va heavy chain (residues 1-506), and a carboxyl-terminal doublet of M(r) = 28/26,000 (residues 507-709) which is cleaved by APC at Arg679 to generate an M(r) = 22/20,000 doublet and an M(r) = 6,000 peptide. No cleavage of the light chain of the human cofactor is observed in the presence or absence of PCPS vesicles following 2 h of incubation with APC. Our data demonstrate that inactivation of human factor V and human factor Va only occurs in the presence of a membrane surface after cleavage at Arg306. However, while this cleavage site is exposed on membrane-bound human factor V, cleavage at Arg506 on the heavy chain of factor Va appears necessary for complete exposure of the cleavage site at Arg306.

Amino Acid Sequence↗
PubMed1994

Osteonectin in matrix remodeling. A plasminogen-osteonectin-collagen complex.

Osteonectin is an adhesive glycoprotein synthesized constitutively by osteoblasts, endothelial cells, and megakaryocytes. Bone-derived and platelet-derived osteonectins differ in their electrophoretic mobility and carbohydrate content, and each displays different affinities for collagen matrices. Both types of osteonectin bind to plasminogen (Kd(app), of 4.7 +/- 1.0 x 10(-8) M for bone osteonectin and 1.2 +/- 0.1 x 10(-7) M for platelet osteonectin). The osteonectin-plasminogen interaction is inhibited by alpha 2-antiplasmin and epsilon-aminocaproic acid, suggesting that the interaction is mediated through the kringle 1 region of plasminogen. Both osteonectins enhance the rate of plasmin generation by tissue-type plasminogen activator to approximately the same extent as fibrinogen. Equilibrium binding measurements conducted using total internal reflection fluorescence spectroscopy indicate that plasminogen binds to collagen in the presence of bone osteonectin (Kd = 1.30 +/- 0.1 x 10(-7) M). No binding of plasminogen to collagen matrix was detected in the presence of platelet osteonectin or in the absence of bone osteonectin. Bone osteonectin-dependent binding of plasminogen to collagen matrix is reversed by the addition of epsilon-aminocaproic acid. The ability of both types of osteonectin to bind to and influence plasminogen activation and of bone osteonectin to anchor plasminogen on collagen matrices suggests that osteonectin may play a role in directing extracellular matrix proteolysis.

Aminocaproic Acid↗
PubMed1994

Determination of the disulfide bridges in factor Va heavy chain.

The M(r) = 94,000 heavy chain of bovine factor Va contains 10 cysteine residues which are distributed in the 2 A domains which make up this portion of the factor V molecule. The A1 domain contains four cysteines while the A2 domain contains six cysteines. The locations of disulfide bridges and free cysteines in bovine factor Va heavy chain were analyzed using iodo[14C]acetamide-labeled factor Va heavy chain digested with trypsin, plasmin, V-8 protease, and cyanogen bromide. Following HPLC separation of the resulting peptides, free cysteines were identified by the incorporation of radioactivity while disulfide-containing peptides were detected using an SBD-F fluorometric assay after reduction. All cysteine-containing peptides were analyzed by amino acid sequence analysis. The four cysteines in the A1 domain are associated with two disulfide bonds, Cys139-Cys165 and Cys220-Cys301. One disulfide bond was explicitly identified in the A2 domain; Cys471-Cys497, and a free cysteine was found in the A2 domain at Cys538. Significant difficulties were encountered in preparing identifiable or soluble peptides which would permit the explicit identification of the three remaining cysteines in the A2 domain. On the basis of homology, it is likely that Cys589 is a free SH while a disulfide bridge exists between Cys579 and Cys660. Thus, three major disulfide bonding patterns, characterized as "alpha", "beta", and "gamma" loops, are found in factor V. Each A domain contains a 26 residue "alpha loop at positions 139-165, 471-497, and 1684-1710. The A1 and A2 domains each contain 81 amino acid residue "beta" loops at 220-301 and 579-660.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

A membrane-mediated catalytic event in prothrombin activation.

Prothrombinase assembly takes place on the surface of unsaturated phosphatidylcholine (PC), phosphatidylserine (PS) membranes in the presence of Ca2+, through the rapid association of membrane-bound factor Va and factor Xa. The present study uses saturated PCPS (75:25, w/w) vesicles to study prothrombinase assembly and catalytic properties in order to differentiate the influences of the membrane upon catalyst assembly, substrate delivery, and peptide bond cleavage. In contrast to studies using unsaturated phospholipid, prothrombin activation studies using saturated PCPS (75:25, w/w) (C14:0, C16:0, and C18:0) revealed up to a 20-fold decrease in prothrombinase activity. C18:0 membranes support at least 50% of the prothrombinase binding capacity (KdVa-Xa = 1 nM and nVa-Xa = 1.1) of C18:1 PCPS (75:25, w/w). Thus, the 95% loss in activity cannot be explained by gross alterations in catalyst concentration or assembly. Stopped-flow studies with saturated lipids demonstrate that factor Va, factor Xa, and prothrombin have decreased kon values. Compensatory changes in koff leave the Kd values for these protein-lipid interactions almost unchanged relative to unsaturated PCPS. The profoundly decreased activation rate on saturated phospholipid membranes as compared to unsaturated phospholipids is in part due to slowed substrate/enzyme delivery caused by the saturated lipids. However, studies using prethrombin-1 and C18:0 PCPS (75:25, w/w) also revealed a 15-fold decrease in activity for preassembled prothrombinase. Although there was a slight change in Km (+2-fold), the major cause of the decrease is an 18-fold decrease in kcat. Similar differences for Km and kcat values were obtained for prothrombin. Substrate delivery is thus only partially responsible for the diminished prothrombinase activity observed with saturated phospholipids. Since the activity of prothrombinase is decreased for both prothrombin and prethrombin-1 principally by reducing kcat, it appears that catalyst formation on saturated phospholipids somehow compromises the proteolytic activity of the enzyme complex. This implies that the phospholipid bilayer serves not merely as a surface for condensing the proteins but also as a functional element of the prothrombinase enzyme.

Amino Acid Sequence↗
PubMed1994

A model for the tissue factor pathway to thrombin. I. An empirical study.

The activation of prothrombin, factor V, factor VIII, factor IX, and factor X by the tissue factor-factor VIIa complex, in vitro, in a system in which each precursor protein was present at plasma concentration, was evaluated using a combination of activity assays, immunoblots, active-site blots, and autoradiography. The thrombin generation curves observed were distinctly nonlinear and typically displayed a time lag in which little or no thrombin was observed. This was followed by an almost linear propagation phase of thrombin formation. The lag was a function of tissue factor/factor VIIa concentration and represented primarily the interval of factor V and factor VIII activation. The postlag propagation phase of thrombin generation was nearly independent of the initial activator (factor VIIa or tissue factor) concentration over a 10(3)-fold range in factor VIIa-tissue factor concentration. Maximum thrombin generation rates were observed when less than 1% of the factor IX and X present was activated but when nearly 100% activation of the cofactors, factor V and factor VIII, was achieved. Analyses of the activation pattern of factor V indicated that the cofactor is activated by both factor Xa and thrombin which are formed at low levels during the lag phase of the reaction. When the initial reaction mixture contained factor Va instead of factor V, the lag was substantially reduced. When factor V was deleted from the reaction mixture, no thrombin formation was observed. When either factor VII or factor IX was deleted from the reaction system, the propagation phase of thrombin formation (at 5 pM tissue factor-factor VIIa complex) was only one-third that observed for reactions which contained factor VIII and factor IX. The addition of factor XI to the experimental system increased the rate of thrombin formation by 15% during the propagation phase but had no effect upon the lag phase of the reaction. Our data suggest that normal hemostasis may be initiated by the factor VIIa-tissue factor complex and support the concept of multiple feedback reactions which amplify and propagate the hemostatic response.

Blood Coagulation Factors↗