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Biomedical subjects

M W Mosesson

Publications and source records attributed to M W Mosesson.

At least 55 records · Page 3Linked to original sources
PubMed1995

Development of a whole platelet ELISA to detect circulating activated platelets.

P-selectin is a granule membrane protein that is expressed on the surface of activated endothelial cells and platelets. Flow cytometry has been used as a means of detecting activated platelets in the circulation by using antibodies to P-selectin and other surface markers. In the study reported here, we developed a whole platelet ELISA for measuring P-selectin on platelets in platelet-rich plasma. Platelet-rich plasma samples for analysis were isolated from fresh blood by centrifugation, fixed with 1.0% paraformaldehyde, and used within 3 hours or after storage at -70 degrees C for up to 10 months. Paraformaldehyde-fixed, phorbol myristate acetate-activated or thrombin receptor peptide-activated platelets were used to construct a standard calibration curve. These platelets were stable after 10 months of storage at -70 degrees C. Interassay variability showed a high degree of correlation, with r = 0.98 +/- 0.03 (n = 12). The accuracy and specificity of the ELISA was verified by using fluorescence-activated flow cytometric analysis and is as sensitive (< or = 0.5%) as flow cytometry for detecting P-selectin expression on platelets. To assess the ability of the platelet ELISA to detect platelet activation in the systemic circulation, we examined 24 patients with unstable angina and 12 age-matched control subjects. Patients with unstable angina demonstrated significantly higher levels of circulating activated platelets than did age-matched control subjects. Although storage-dependent differences in absolute platelet activation levels were found, platelet ELISA results of samples evaluated within either 3 hours or after 10 months of storage were comparable to results obtained by fluorescence-activated flow cytometric analysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Angina, Unstable↗
PubMed1995

Sequence of release of fibrinopeptide A from fibrinogen molecules by thrombin or Atroxin.

During the conversion of fibrinogen to fibrin, two amino-terminal fibrinopeptides A (FPAs) are cleaved by thrombin from each molecule. During early phases of conversion, fibrin intermediates lacking one of two FPAs (des A fibrin) are produced, the level of which depends on whether the FPA cleavage sequence from each molecule is random of concerted. Random cleavage of FPA would produce higher levels of des A fibrin at any thrombin concentration than would concerted cleavage, and the level of this intermediate product would have an important effect on the ultimate structure of the fibrin clot. Because evidence bearing on this subject is conflicting, we carried out experiments to assess the FPA release sequence from fibrinogen by thrombin or by an FPA-cleaving snake venom enzyme, Atroxin. At timed intervals the enzymatic reaction was terminated by precipitation with trichloroacetic acid, and the precipitate was then treated with cyanogen bromide to produce a dimeric amino-terminal fragment. These disulfide-linked amino-terminal fragments of fibrinogen, containing both, one, or neither FPA, were then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and their distribution quantified by densitometry. The rates of cleavage of the first FPA, k1, and of the second FPA, k2, were computed by fitting the data to equations for a consecutive chemical reaction. This analysis indicated that cleavage by either enzyme resulted in substantial amounts of des A fibrin intermediates. The ratio of the cleavage rates (k2/k1) was higher for thrombin (1.2 +/- 0.3) than it was for Atroxin (0.7 +/- 0.2) but indicates in both cases that the release rate of the second FPA is nearly the same as that of the first FPA.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

Orientation of the carboxy-terminal regions of fibrin gamma chain dimers determined from the crosslinked products formed in mixtures of fibrin, fragment D, and factor XIIIa.

There are two schools of thought regarding the orientation of the intermolecular epsilon-amino-(gamma-glutamyl) lysine isopeptide bonds formed between gamma chains in the D domains of assembled fibrin fibers. Some investigators believe that these bonds are oriented parallel to the direction of fiber growth (longitudinally) at the contacting ends of fibrin D domains ('DD-long'), whereas others believe that these bonds are oriented across the two-stranded fibril, between D domains in opposing strands ('DD-transverse'). To distinguish between these two possibilities, the structure of crosslinked products formed in mixtures of fibrin, plasmic fragment D, and factor XIIIa were analyzed, based upon this rationale: Complex formation between D fragments and a fibrin template depends upon the non-covalent 'D:E' interaction between each fibrin E domain and two D fragments ('D:fibrin:D'). If carboxy-terminal gamma chains in the D:fibrin:D complex become aligned in a DD-long configuration, only crosslinked fragment D dimers ('D-D') will result and the fibrin 'template' will not become crosslinked to the associated D fragments. If instead, gamma chain crosslinks form transversely between the D fragments and fibrin, covalently linked D-fibrin complexes will result. SDS-PAGE of factor XIIIa crosslinked mixtures of fibrin and fragment D demonstrated products of a size and subunit composition indicating D-fibrin and D-fibrin-D formation. Small amounts of D dimers were also formed at the same levels as were formed in mixtures of fragment D and factor XIIIa alone. Electron microscopic images of D-fibrin-D complexes prepared under physiological buffer conditions demonstrated that the D fragments were associated with the central E domain of the fibrin molecule, but that they could be dissociated from this non-covalent association in 2% acetic acid. These findings indicate that gamma chain crosslinks occur transversely in D:fibrin:D complexes and permit the extrapolated conclusion that gamma chain crosslinks are also positioned transversely in an assembled fibrin polymer.

Fibrin↗
PubMed1994

Progressive cross-linking of fibrin gamma chains increases resistance to fibrinolysis.

In the presence of plasma transglutaminase (factor XIIIa) fibrin first undergoes intermolecular covalent cross-linking between its gamma chains to create gamma dimers followed by slower cross-linking among its alpha chains to form alpha polymers. Progressive cross-linking of gamma chain dimers occurs at the slowest rate, resulting in gamma trimers and gamma tetramers ("gamma multimers"). Most studies indicate that cross-linked fibrin clots become resistant to fibrinolysis, but the basis for this event is not clear. In this study, we explored the role of gamma chain multimerization compared with alpha polymerization as causal factors in time-dependent development of resistance to fibrinolysis. Fibrin clots prepared from native (intact) fibrinogen were incubated for up to 120 h at near physiological ionic strength and a factor XIIIa level approximating that in plasma. These clots were lysed by plasmin at rates that were inversely proportional to the level of gamma multimers, which increased progressively with the time of incubation. In contrast, fibrin cross-linked at high ionic strength (a condition under which only gamma dimers and alpha polymers form) or fibrin formed in the absence of factor XIII showed no time-dependent decrease in lysis rates. Fibrin cross-linked for a fixed time period with increasing amounts of factor XIIIa contained gamma multimer levels that were proportional to the factor XIIIa concentration and lysed at rates that were inversely proportional to the gamma multimer level. Furthermore, cross-linked fibrin formed from fibrinogen fraction I-9, which has limited potential for alpha polymerization, showed the same reduction in the lysis rate as native cross-linked fibrin. These findings indicate that development of resistance to fibrinolysis of cross-linked fibrin is not measurably dependent upon gamma dimer or alpha polymer formation but develops solely as a function of gamma multimerization.

Chromatography, Affinity↗
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↗