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M W Mosesson

Publications and source records attributed to M W Mosesson.

At least 37 records · Page 2Linked to original sources
PubMed1993

Evidence for a second type of fibril branch point in fibrin polymer networks, the trimolecular junction.

Fibrin molecules polymerize to double-stranded fibrils by intermolecular end-to-middle domain pairing of complementary polymerization sites, accompanied by fibril branching to form a clot network. Mass/length measurements on scanning transmission electron microscopic images of fibrils comprising branch points showed two types of junctions. Tetramolecular junctions occur when two fibrils converge, creating a third branch with twice the mass/length of its constituents. Newly recognized trimolecular junctions have three fibril branches of equal mass/length, and occur when an extraneous fibrin molecule initiates branching in a propagating fibril by bridging across two unpaired complementary polymerization sites. When trimolecular junctions predominate, clots exhibit nearly perfect elasticity.

Blood Coagulation↗
PubMed1993

Comparison of the sequence of fibrinopeptide A cleavage from fibrinogen fragment E by thrombin, atroxin, or batroxobin.

In order to investigate the sequence of fibrinopeptide release from the amino terminal end of a dimeric fibrinogen-derived substrate by thrombin or batroxobins, we studied their effects on plasmic fragment E1, a core fragment from the central domain of fibrinogen containing both A alpha chain fibrinopeptide A (FPA) sequences. Isoelectric focussing (IEF) was employed as a means of resolving des A-fragment E1, from which one FPA had been cleaved, from des AA-fragment E1 resulting from the loss of both FPA's. Using densitometric gel scanning for quantification of the levels of intact fragment E1, des A-fragment E1, and des AA-fragment E1, in mixtures incubated with enzyme for various periods of time, we found similar catalytic rate constants (k1, k2) for release of the first fibrinopeptide A, (FPA1) or the second, (FPA2) from fragment E1, with either thrombin or batroxobin (k2:k1 ratios of 1.10 +/- 0.42, 1.34 +/- 0.26 respectively). Atroxin released FPA2 more slowly than FPA1 with a k2:k1 ratio of 0.34 +/- 0.1. Th finding that the cleavage of FPA2 by Atroxin is three-fold slower than thrombin and almost four-fold slower than batroxobin, suggest that batroxobin and thrombin cleavage of FPA2 may be cooperative in nature. However, the cooperativity in the cleavage sequence is insufficient to markedly suppress the evolution of intermediate des A fragment E species during early and intermediate phases of FPA cleavage from fragment E.

Amino Acid Sequence↗
PubMed1993

Paris I dysfibrinogenemia: a point mutation in intron 8 results in insertion of a 15 amino acid sequence in the fibrinogen gamma-chain.

Paris I dysfibrinogenemia results in the production of a fibrinogen molecule containing a functionally abnormal gamma-chain. We determined the basis of the molecular defect using polymerase chain reaction (PCR) to amplify the gamma-chain region of the Paris I subject's genomic DNA. Comparative sequence analysis of cloned PCR segments of normal and Paris I genomic DNA revealed only an A-->G point mutation occurring at nucleotide position 6588 within intron 8 of the Paris I gamma-chain gene. We examined six normal individuals and found only normal sequence in this region, indicating that this change is not likely to represent a normal polymorphism. This nucleotide change leads to a 45 bp fragment being inserted between exons 8 and 9 in the mature gamma Paris I chain mRNA, and encodes a 15 amino acid insert after gamma 350 [M-C-G-E-A-L-P-M-L-K-D-P-C-Y]. Alternative splicing of this region from intron 8 into the mature Paris I gamma-chain mRNA also results after translation into a substitution of S for G at position gamma 351. Biochemical studies of 14C-iodoacetamide incorporation into disulfide-reduced Paris I and normal fibrinogen corroborated the molecular biologic predictions that two additional cysteine residues exist within the gamma Paris I chain. We conclude that the insertion of this amino acid sequence leads to a conformationally-altered, and dysfunctional gamma-chain in Paris I fibrinogen.

Afibrinogenemia↗
PubMed1993

Molecular basis for fibrinogen Dusart (A alpha 554 Arg-->Cys) and its association with abnormal fibrin polymerization and thrombophilia.

The molecular defect in the abnormal fibrinogen Dusart (Paris V) that is associated with thrombophilia was determined by sequence analysis of genomic DNA that had been amplified using the polymerase chain reaction. The propositus was heterozygous for a single base change (C-->T) in the A alpha-chain gene, resulting in the amino acid substitution A alpha 554 Arg-->Cys. Restriction analysis of the amplified DNA derived from the family members showed that his father and his two sons were also heterozygous. Electron microscopic studies on fibrin formed from purified fibrinogen Dusart demonstrated fibers that were much thinner than in normal fibrin. In contrast to the previously observed defective binding of plasminogen, the binding of thrombospondin to immobilized fibrinogen Dusart was similar to that of normal fibrinogen. Immunoblot analysis of plasma fibrinogen demonstrated that a substantial part of the fibrinogen Dusart molecules were disulfide-linked to albumin. The plasma of the affected family members also contained fibrinogen-albumin complexes. Furthermore, small amounts of high molecular weight complexes containing fibrinogen were detected in all the heterozygous individuals. These data indicate that the molecular abnormality in fibrinogen Dusart (A alpha 554 Arg-->Cys) results in defective lateral association of the fibrin fibers and disulfide-linked complex formation with albumin, and is associated with a family history of recurrent thrombosis in the affected individuals.

Adult↗
PubMed1993

The cleavage sequence of fibrinopeptide A from fibrinogen fragment E by thrombin, atroxin or batroxobin.

Calculations of data from fibrin polymerization and cross-linking experiments infer that thrombin-catalysed release of the second of the two fibrinopeptides A (FpA2) from fibrinogen is concerted, although other data suggest that FpA2 release is random. In the concerted pattern of FpA release, divalent monomer (des AA-fibrin) formation predominates throughout the enzymatic conversion of fibrinogen to fibrin, an effect leading to relatively rapid fibril assembly. Alternatively, random FpA2 release would result in a substantial population of monovalent monomer (des A-fibrin) intermediates during early and intermediate phases of the enzymatic conversion to fibrin. Their formation would cause a delay in fibrin fibril assembly. In order to address the question of the pattern of FpA release directly, we purified plasmic fibrinogen fragment E1 isoforms containing both FpA sequences and studied the sequence of FpA release by thrombin or batroxobin. Des A-fragment E1 intermediates formed by loss of one FpA (FpA1), and des AA-fragment E1 products (lacking both FpA1 and FpA2) were identified by analytical isoelectric focusing and quantified by densitometry. The catalytic rate of release of FpA1 (k1) and FpA2 (k2) by thrombin or batroxobin was similar. The ratio of these rates, k2:k1, was 1.10 +/- 0.42 for thrombin and 1.34 +/- 0.26 for batroxobin. These findings indicate that these enzymes cleave FpA2 randomly from fragment E1.

Batroxobin↗
PubMed1993

The polymerization of fibrinogen Dusart (A alpha 554 Arg-->Cys) after removal of carboxy terminal regions of the A alpha-chains.

The six polypeptide chains of normal fibrinogen are covalently linked by interchain disulphide bonds, and there are no free sulphydryl groups. Fibrinogen Dusart is a congenital fibrinogen variant in which A alpha 554 Arg is replaced by Cys; albumin is disulphide linked to these fibrinogen molecules, possibly at A alpha 554 Cys. Functionally, Dusart fibrinogen displays markedly abnormal fibrin polymerization, characterized by delayed lateral fibril association and matrix fibre bundles that are thinner than normal fibrin bundles. These observations are consistent with experiments suggesting that the carboxy terminal region of the A alpha-chain contains a polymerization domain that participates in lateral fibril associations. In order to investigate the location and the effect of albumin binding to Dusart fibrinogen, we examined the fibrinogen by electron microscopy, and compared the polymerization and ultrastructure of fibrin prepared from normal fibrinogen containing intact A alpha-chains (fraction I-2) or plasmin degraded fibrinogen molecules lacking carboxy terminal regions of A alpha-chains (fraction I-9D), with fibrin prepared from Dusart fraction I-2 and I-9D. Most bound albumin was released from Dusart fibrinogen by plasmin degradation involving the A alpha-chains. Nevertheless, we were able to visualize albumin molecules remaining covalently bound to Dusart I-9D as well as to Dusart I-2 fibrinogen, as distinct globular domains situated near the fibrinogen D domain. The presence of albumin in these fractions was confirmed by Western blotting using anti-albumin. Dusart fibrin polymerized much more slowly than normal I-2, as previously reported, whereas polymerization of Dusart I-9D fibrin was faster than Dusart I-2 and nearly the same as normal I-9D fibrin.(ABSTRACT TRUNCATED AT 250 WORDS)

Albumins↗
PubMed1992

Characterization of the gamma chain platelet binding site on fibrinogen fragment D.

Glycoprotein (GP) IIb/IIIa on adenosine diphosphate (ADP)-activated human platelets interacts with specific sites on the fibrinogen molecule leading to aggregation. We characterized the platelet-binding site on the gamma chains of fibrinogen using plasmic fragments D gamma A and D gamma'. Fragment D gamma A, which contains the carboxy terminal gamma A400-411 platelet-binding sequence (HHLGGAKQAGDV), was 70-fold more active than the synthetic gamma A400-411 peptide in inhibiting ADP-induced platelet aggregation. Fragment D gamma A inhibited fibrinogen binding and also bound directly to ADP-activated platelets. The Kd values determined for fibrinogen and fragment D gamma A binding were 0.55 mumol/L and 1.2 mumol/L, respectively. In contrast, fragment D gamma', which differs from fragment D gamma A with respect to its gamma chain sequence from position 408 to the COOH-terminus at position 427, did not inhibit platelet aggregation or fibrinogen binding, and did not bind directly to the platelet surface. Denaturation of fragment D gamma A with guanidine-HCl caused a loss of inhibitory activity in platelet aggregation assays. These data indicate that the native conformation of the gamma chain platelet-binding site on fibrinogen is important for optimal binding to GPIIb/IIIa.

Adenosine Diphosphate↗
PubMed1992

Factors affecting gamma-chain multimer formation in cross-linked fibrin.

The major covalently linked multimolecular D fragments found in plasmic digests of factor XIIIa cross-linked fibrin formed under physiological pH and ionic strength conditions consist of D dimers, D trimers, and D tetramers. These fragments are linked by epsilon-amino-gamma-glutamyllysine bonds in the carboxy-terminal regions of their gamma chains, which had originated in the cross-linked fibrin as gamma dimers, gamma trimers, and gamma tetramers, respectively. In this study, factors affecting the degree and rate of formation of these three classes of cross-linked gamma chains were determined by analyzing the D-fragment content of plasmic digests of cross-linked fibrin that had been sampled after all gamma-chain monomers had been consumed in the cross-linking process. D trimers and D tetramers, expressed as a proportion of the total D-fragment content, both increased at the expense of the D-dimer population as a function of increasing factor XIII concentration, the time of cross-linking, or the CaCl2 concentration. Their levels decreased as the ionic strength was raised by NaCl addition. However, the ionic strength effect could be reversed by concomitantly raising the CaCl2 concentration. Digests of clots prepared from recalcified fresh citrated plasma also contained each type of cross-linked D fragment, and the proportion of D trimers and D tetramers in the digest increased with increasing clot incubation time. These results indicate that gamma-trimer and gamma-tetramer formation is a dynamic physiological process.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium↗
PubMed1992

The assembly and structure of the fibrin clot.

This review summarizes our current understanding of the structure of the fibrinogen molecule, its conversion to fibrin, and the main features of fibrin clot assembly and crosslinking. Several major aspects of fibrin matrix assembly and crosslinking are newly discovered, and differ significantly from more traditional views of fibrin structure. The new information that I have summarized will be of importance for correctly understanding the role that fibrin plays in the hemostatic process, and for learning how the structure of the fibrin clot network contributes to its strength, elasticity, interactions with cells, tissue matrix, and its susceptibility to fibrinolysis.

Amino Acid Sequence↗
PubMed1992

The roles of fibrinogen and fibrin in hemostasis and thrombosis.

Proteolytic conversion of fibrinogen to fibrin results in self-assembly to form a clot matrix that subsequently becomes cross-linked by fXIIIa to form the main structural element of the thrombus in vivo. Fibrin formation and assembly lead to new properties that regulate the rate and extent of clotting, cross-linking, and fibrinolysis. These are brought about by the ability of fibrin (1) to bind thrombin at a nonsubstrate site, thus limiting its diffusability but at the same time preserving its catalytic potential; (2) to bind fXIII, regulate its activation to fXIIIa, and limit further activation of fXIII once fibrin cross-linking has occurred; and (3) to bind alpha 2-PI, t-PA, and plasminogen and regulate the initiation and propagation of fibrinolysis. Fibrinogen and fibrin contain several potential platelet binding sites that interact with platelet GPIIb/IIIa receptors, and thus promote their participation in the hemostatic process. Additional, less well-defined interactions, not covered in detail here, such as those between fibrinogen or fibrin and other plasma proteins, cells, or tissue matrix components, suggest other functions that, along with those detailed above, will further define its multiple roles in modulating hemostasis, inflammation, and the wound healing process.

Amino Acid Sequence↗
PubMed1991

Ionic-strength- and pH-dependent conformational states of human plasma fibronectin.

In order to provide a more detailed understanding of human plasma fibronectin (PFn) solution structure, we examined the effects of pH and ionic strength (mu) variation on the sedimentation velocities (s20,w), fluorescence polarization-derived mean harmonic rotational relaxation times (rho H), far-ultraviolet (UV) circular dichroism (CD), and intrinsic tryptophan fluorescence of dimeric PFn and the monomeric 190/170-kDa PFn fragment. By comparing the biophysical properties of PFn with those of the 190/170-kDa PFn fragment, we could assess the relative importance of intrasubunit and intersubunit electrostatic forces in the stabilization of PFn structure. The rho H derived from isothermal polarization measurements on 1-pyrenebutyrate conjugated PFn decreased markedly (4.5----1.05-1.23 microseconds) when mu was increased from 0.2 to 1.2 or when the pH was adjusted from 7.4 to 2.0 or 11.0. We also noted a significant decrease in the PFn s20,w (13----8.5-9.6S) under these same solvent conditions. In contrast, the rho H and s20,w of the monomeric 190/170-kDa PFn fragment were relatively insensitive to changes in mu or pH. Computer simulations of the observed pH-dependent changes in the far-UV CD of PFn and the 190/170-kDa PFn fragment revealed only minor differences in protein secondary structure. We also observed only small bathochromic shifts (1-3 nm) in the emission maxima of PFn and 190/170-kDa PFn fragment tryptophan fluorescence under acidic or high mu conditions. These results suggest that minimal changes in PFn tertiary (i.e., intrasubunit) structure occur at pH 2, 11, or at mu = 1.2.(ABSTRACT TRUNCATED AT 250 WORDS)

Circular Dichroism↗
PubMed1991

Evidence for thrombin enhancement of fibrin polymerization that is independent of its catalytic activity.

Inhibition of thrombin proteolysis of fibrinogen with D-phenylalanyl-L-propyl-L-arginine chloromethyl ketone (PPACK) results in irreversible inactivation of the thrombin catalytic site, but the PPACK-inhibited thrombin, through its exosite, retains its ability to bind to fibrinogen or fibrin. Hirudin inactivates thrombin at the catalytic site and also inhibits thrombin exosite binding to fibrin or fibrinogen. PPACK or hirudin was added to a clotting mixture of fibrinogen and active thrombin (enzyme-to-substrate ratio = 1:400 at ionic strength of 0.14; 1:800 at ionic strength of 0.09) before the onset of gelation. Subsequent fibrin assembly was evaluated by turbidity measurements at 350 nm and by determining the fibrin and fibrinogen content of the clots that ultimately formed. Polymerization rates and the fibrin-fibrinogen content of the clots that formed were greater in the PPACK-inhibited system than in the hirudin-inhibited system, and the effect was amplified at the lower ionic strength. PPACK-thrombin also promoted the polymerization of native or prepared mixtures of fibrin and fibrinogen. The results suggest that in addition to its well-recognized role in the proteolytic conversion of fibrinogen to fibrin, thrombin functions through exosite binding to fibrin as a cofactor in fibrin polymerization by accelerating fibrin clot assembly.

Amino Acid Chloromethyl Ketones↗
PubMed1990

The polymerization and thrombin-binding properties of des-(B beta 1-42)-fibrin.

Multiple factors affect the thrombin-catalyzed conversion of fibrinogen to fibrin, including: fibrinopeptide (FPA and FPB) release leading to exposure of two types of polymerization domains ("A" and "B," respectively) in the central portion of the molecule, and exposure of a noncatalytic "secondary" thrombin-binding site in fibrin. Fibrinogen containing the FPA sequence but lacking the B beta 1-42 sequence ("des-(B beta 1-42)-fibrinogen"), was compared to native fibrinogen (containing both FPA and FPB) to investigate the role played by B beta 1-42 in the polymerization of alpha-fibrin (i.e. fibrin lacking FPA), to compare reptilase and thrombin cleavage of FPA from fibrinogen, and to explore the location and function of the secondary thrombin-binding site. Electron microscopy of evolving polymer structures (mu, 0.14; pH 7.4) plus turbidity measurements, showed that early thin fibril formation as well as subsequent lateral fibril associations were impaired in des-(B beta 1-42)-alpha-fibrin, thus indicating that the B beta 1-42 sequence contributes to the A polymerization site. Reptilase-activated des-(B beta 1-42)-alpha-fibrin polymerized even more slowly than thrombin-activated des-(B beta 1-42)-alpha-fibrin, differences that disappeared when repolymerization of preformed fibrin monomers was carried out. Since existing data indicate that thrombin releases FPA in a concerted manner, resulting in relatively rapid evolution of fully functional divalent alpha-fibrin monomers, it can be inferred that delayed fibrin assembly of reptilase fibrin is due to slower formation of divalent alpha-fibrin monomers. Thrombin-activated des-(B beta 1-42)-alpha-fibrin polymerized more rapidly at low ionic strength (mu, 0.04) than did native alpha,beta-fibrin, a reversal of their behavior at physiological ionic strength (mu, 0.14). Concomitant measurement of FPA release revealed modest slowing of release at low ionic strength from des-(B beta 1-42)-fibrinogen (t1/2, 36.5 versus 21.5 min) and marked slowing from native fibrinogen (t1/2, 138 versus 22.2 min). This behavior correlated with increased thrombin binding to native alpha,beta-fibrin at low ionic strength, coupled with weak thrombin binding to des-(B beta 1-42)-alpha-fibrin, and indicates that secondary thrombin binding plays an important role in regulating thrombin diffusion and catalytic activity. Des-(B beta 1-42)-fibrinogen lacks or has a markedly defective secondary thrombin-binding site, from which we conclude that the B beta 15-42 sequence in fibrin plays a major role in forming or providing this site.

Batroxobin↗
PubMed1990

Structural model of factors V and Va based on scanning transmission electron microscope images and mass analysis.

Coagulation factor V (fV) is a single-chain glycoprotein (Mr 330,000; domain structure A1-A2-B-A3-C1-C2) that is activated to factor Va (fVa; Mr 174,000) by thrombin, which cleaves away the B domain leaving a heterodimeric structure composed of a heavy chain (A1-A2; Mr 94,000) and a light chain (A3-C1-C2; Mr 74,000). We analyzed the ultrastructure of scanning transmission electron microscope images of bovine and human fV, bovine fVa, and its constituent light chains and heavy chains. Factor V molecules had irregularly globular (10-12 nm) to oblong (8-14 nm) core structures which commonly displayed a peripheral satellite appendage of variable morphology attached to the core by a narrow stalk. Scanning transmission electron microscope mass analyses indicated that monomolecular bovine fV molecules had a mass of 322 +/- 45 kDa and human fV, 315 +/- 31 kDa. Factor Va molecules were irregular, globular (8-12 nm) structures that resembled the fV core structure, lacked the satellite appendage representing B domainal structures, and had a mass of 180 +/- 22 kDa. Our findings permit us to propose a structural model of fV suggesting the relative orientation of its closely associated light chain and heavy chain core components and indicating that these constituents remain associated in the transition from fV to fVa.

Animals↗
PubMed1990

Human plasma fibronectin structure probed by steady-state fluorescence polarization: evidence for a rigid oblate structure.

In order to more clearly define the structure of human plasma fibronectin (PFn) under physiologic buffer conditions, we determined the mean harmonic rotational relaxation times (rho H) of PFn and the thrombin-derived 190/170-kDa PFn fragment using steady-state fluorescence polarization. These measurements utilized the long lifetime emission (tau = 1.2 X 10(-7) S) exhibited by 1-pyrenebutyrate, which had been covalently attached to amino groups at random sites on the PFn subunit. Our data analysis assumed that two independent processes depolarize the fluorescence exhibited by the dansylcadaverine and 1-pyrenebutyrate conjugates of PFn: (A) rapid (rho H less than 10(-9) S) "thermally-activated" localized rotational motion of the protein side chains bearing the fluorescent probe [Weber, G. (1952) Biochem. J. 51, 145-154] and (B) slow (rho H approximately 10(-6) S) temperature-independent global rotational motion of the whole PFn molecule. Since only the rho H associated with the latter process is a true hydrodynamic parameter (i.e., sensitive to size and/or shape of the PFn molecule), we utilized isothermal polarization measurements to discriminate against the interfering signal arising from "thermally activated" probe rotation. The rho H (4.4 +/- 0.9 microseconds) derived from an experiment in which pyrene-PFn fluorescence polarization was monitored as a function of sucrose concentration at constant temperature is 7 (+/- 1.4) times longer than that predicted for an equivalent hydrated sphere. We propose that "thermally activated" probe rotation gives rise to the nearly 100-fold shorter PFn rho H values previously reported in the literature. Consequently, our data exclude all previous models which invoke segmental flexibility of the PFn peptide backbone. The simplest hydrodynamic model supported by our fluorescence data is an oblate ellipsoid with an axial ratio of 15:1. All prolate models can be unambiguously excluded by this result. We estimate that the disk-shaped PFn molecule has a diameter and thickness of 30 and 2 nm, respectively. Electron microscopy of negatively stained PFn specimens on carbon also showed PFn to have a compact rounded structure. The much faster rotational relaxation rate of the pyrene-190/170-kDa PFn fragment (rho H = 0.92 +/- 0.11 microseconds) compared to pyrene-PFn indicated that this monomeric PFn fragment, like native PFn, had an oblate shape under physiologic buffer conditions.

Chromatography, Affinity↗
PubMed1990

The acceleratory effect of thrombin on fibrin clot assembly.

Inhibition of thrombin proteolysis of fibrinogen with D-phenylalanyl-L-propyl-L-arginine chloromethyl ketone (PPACK) results in irreversible inactivation of the thrombin catalytic site, but the PPACK-inhibited thrombin, through its exosite, retains its ability to bind to fibrinogen or fibrin. Hirudin inactivates thrombin at the catalytic site and also inhibits thrombin exosite binding to fibrin or fibrinogen. PPACK or hirudin was added to a clotting mixture of fibrinogen and active thrombin (enzyme:substrate ratio, 1:400 and 1:800) prior to the onset of gelation. Subsequent fibrin assembly was evaluated by turbidity measurements at 350 nm and by determining the fibrin and fibrinogen content of the clots that ultimately formed. Polymerization rates and the fibrin/fibrinogen content of the clots that formed were greater in the PPACK-inhibited system than in the hirudin-inhibited system. Lowering the ionic strength from 0.14 to 0.09 amplified these differences. The results suggest that in addition to its well-recognized role in the proteolytic conversion of fibrinogen to fibrin, thrombin functions as a cofactor in the fibrin assembly process.

Amino Acid Chloromethyl Ketones↗