Role of Src in the Modulation of Multiple Adaptor Proteins in FcRI Oxidant Signaling

Blood ◽  
1999 ◽  
Vol 94 (6) ◽  
pp. 2112-2120 ◽  
Author(s):  
Rae-Kil Park ◽  
Kayvon D. Izadi ◽  
Yashwant M. Deo ◽  
Donald L. Durden
Keyword(s):  
Tyrosine Phosphorylation ◽  
Kinase Inhibitor ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Src Kinase ◽  
Myeloid Cells ◽  
Adaptor Protein ◽  
Cross Linking ◽  
Γ Subunit ◽  
Stimulation Of

Abstract Cross-linking of Fc receptors for IgA, FcR (CD89), on monocytes/macrophages is known to enhance phagocytic activity and generation of oxygen free radicals. We provide evidence here that the FcR signals through the γ subunit of FcɛRI in U937 cells differentiated with interferon γ (IFNγ). Our results provide the first evidence that FcR-mediated signals modulate a multimolecular adaptor protein complex containing Grb2, Shc, SHIP, CrkL, Cbl, and SLP-76. Cross-linking of FcRI using anti-FcRI induces the phosphorylation of the γ subunit as detected by mobility retardation on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Stimulation of FcRI induced the tyrosine phosphorylation of Shc and increased the association of Grb2 with Shc and CrkL. Grb2 associates constitutively with Sos, and the latter undergoes mobility shift upon FcRI stimulation. The complex adapter proteins, Cbl and SLP-76, are physically associated in myeloid cells and both proteins undergo tyrosine phosphorylation upon FcR stimulation. These data indicate that the stimulation of FcR results in the modulation of adaptor complexes containing tyrosine-phosphorylated Cbl, Shc, SHIP, Grb2, and Crkl. Experiments performed with the Src kinase inhibitor, PP1, provide the first evidence that Src kinase activation is required for FcRI-induced production of superoxide anions and provide insight into the mechanism for FcR-mediated activation of downstream oxidant signaling in myeloid cells.

Role of Src in the Modulation of Multiple Adaptor Proteins in FcRI Oxidant Signaling

Blood ◽  
1999 ◽  
Vol 94 (6) ◽  
pp. 2112-2120 ◽  
Author(s):  
Rae-Kil Park ◽  
Kayvon D. Izadi ◽  
Yashwant M. Deo ◽  
Donald L. Durden
Keyword(s):  
Tyrosine Phosphorylation ◽  
Kinase Inhibitor ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Src Kinase ◽  
Myeloid Cells ◽  
Adaptor Protein ◽  
Cross Linking ◽  
Γ Subunit ◽  
Stimulation Of

Cross-linking of Fc receptors for IgA, FcR (CD89), on monocytes/macrophages is known to enhance phagocytic activity and generation of oxygen free radicals. We provide evidence here that the FcR signals through the γ subunit of FcɛRI in U937 cells differentiated with interferon γ (IFNγ). Our results provide the first evidence that FcR-mediated signals modulate a multimolecular adaptor protein complex containing Grb2, Shc, SHIP, CrkL, Cbl, and SLP-76. Cross-linking of FcRI using anti-FcRI induces the phosphorylation of the γ subunit as detected by mobility retardation on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Stimulation of FcRI induced the tyrosine phosphorylation of Shc and increased the association of Grb2 with Shc and CrkL. Grb2 associates constitutively with Sos, and the latter undergoes mobility shift upon FcRI stimulation. The complex adapter proteins, Cbl and SLP-76, are physically associated in myeloid cells and both proteins undergo tyrosine phosphorylation upon FcR stimulation. These data indicate that the stimulation of FcR results in the modulation of adaptor complexes containing tyrosine-phosphorylated Cbl, Shc, SHIP, Grb2, and Crkl. Experiments performed with the Src kinase inhibitor, PP1, provide the first evidence that Src kinase activation is required for FcRI-induced production of superoxide anions and provide insight into the mechanism for FcR-mediated activation of downstream oxidant signaling in myeloid cells.

scholarly journals Fc gamma R-dependent mitogen-activated protein kinase activation in leukocytes: a common signal transduction event necessary for expression of TNF-alpha and early activation genes.

1996 ◽  
Vol 184 (3) ◽  
pp. 1027-1035 ◽  
Author(s):  
R Trotta ◽  
P Kanakaraj ◽  
B Perussia
Keyword(s):  
Nk Cells ◽  
Cell Line ◽  
Kinase Inhibitor ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Mitogen Activated Protein Kinase ◽  
Monocytic Cell ◽  
Fast Kinetics ◽  
Mitogen Activated Protein ◽  
Stimulation Of

Cross-linking the receptors for the Fc domain of IgG (Fc gamma R) on leukocytes induces activation of protein tyrosine kinases. The intermediary molecules that transduce to the nucleus the signals leading to induction of the diverse biological responses mediated by these receptors are not clearly identified. We have investigated whether mitogen-activated protein kinases (MAPK) are involved in transmembrane signaling via the three Fc gamma R present on monocytic, polymorphonuclear, and natural killer (NK) cells. Our results indicate that occupancy of Fc gamma RI and Fc gamma RII on the monocytic cell line THP-I and on polymorphonuclear leukocytes (PMN) induces, transiently and with fast kinetics, MAPK phosphorylation, as indicated by decreased electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and increased amounts of the proteins in antiphosphotyrosine antibody immunoprecipitates. This, associated with increased enzymatic activity, also occurs upon stimulation of the transmembrane isoform of CD16 (Fc gamma RIIIA) in NK cells and in a T cell line expressing transfected Fc gamma RIIIA alpha ligand-binding chain in association with zeta, but not upon stimulation of the glycosil-phosphatidylinositol-anchored Fc gamma RIIIB on PMN. Using the specific MAP kinase kinase inhibitor-PD 098059, we show that activation of MAPK is necessary for the Fc gamma R-dependent induction of c-fos and tumor necrosis factor alpha mRNA expression in monocytes and NK cells. These results underscore the role of MAPK as signal-transducing molecules controlling the expression of different genes relevant to leukocyte biology upon Fc gamma R stimulation.

Stromal cell–derived factor-1α/CXCL12–induced chemotaxis of T cells involves activation of the RasGAP-associated docking protein p62Dok-1

Blood ◽  
2005 ◽  
Vol 105 (2) ◽  
pp. 474-480 ◽  
Author(s):  
Seiichi Okabe ◽  
Seiji Fukuda ◽  
Young-June Kim ◽  
Masaru Niki ◽  
Louis M. Pelus ◽  
...  
Keyword(s):  
T Cells ◽  
Tyrosine Phosphorylation ◽  
Intracellular Signaling ◽  
Stromal Cell ◽  
Kinase Inhibitor ◽  
Src Kinase ◽  
Adaptor Protein ◽  
Mitogen Activated Protein ◽  
Associated Proteins ◽  
Stromal Cell Derived Factor

Abstract Events mediating stromal cell–derived factor-1 (SDF-1α/CXCL12) chemotaxis of lymphocytes are not completely known. We evaluated intracellular signaling through RasGAP-associated protein p62Dok-1 (downstream of tyrosine kinase [Dok-1]) and associated proteins. SDF-1α/CXCL12 stimulated Dok-1 tyrosine phosphorylation and association with RasGAP, adaptor protein p46Nck, and Crk-L in Jurkat T cells. The phosphorylation of Dok-1 was blocked by pretreatment of cells with the src kinase inhibitor PP2. Src kinase family member Lck was implicated. SDF-1α/CXCL12 did not phosphorylate Dok-1 in J.CaM1.6 cells, a Jurkat derivative not expressing Lck, but did phosphorylate Dok-1 in J.CaM1.6 cells expressing Lck. SDF-1α/CXCL12 induced the tyrosine phosphorylation of Pyk2 and the association of Pyk2 with zeta chain–associated protein-70 kilodaltons (Zap-70) and Vav. SDF-1α/CXCL12 enhanced the association of RasGAP with Pyk2. CXCR4–expressing NIH3T3 and Baf3 cells transfected with full-length Dok-1 cDNA were suppressed in their responses to SDF-1α/CXCL12–induced chemotaxis; mitogen-activated protein (MAP) kinase activity was also decreased. Chemotaxis to SDF-1/CXCL12 was significantly enhanced in Dok-1–/– CD4+ and CD8+ splenic T cells. These results implicate Dok-1, Nck, Crk-L, and Src kinases—especially Lck, Pyk2, Zap-70, Vav, and Ras-GAP—in intracellular signaling by SDF-1α/CXCL12, and they suggest that Dok-1 plays an important role in SDF-1α/CXCL12–induced chemotaxis in T cells.

scholarly journals Recruitment of the cross-linked opsonic receptor CD32A (FcγRIIA) to high-density detergent-resistant membrane domains in human neutrophils

2004 ◽  
Vol 381 (3) ◽  
pp. 919-928 ◽  
Author(s):  
Emmanuelle ROLLET-LABELLE ◽  
Sébastien MAROIS ◽  
Kathy BARBEAU ◽  
Stephen E. MALAWISTA ◽  
Paul H. NACCACHE
Keyword(s):  
Tyrosine Phosphorylation ◽  
Plasma Membranes ◽  
Kinase Inhibitor ◽  
Src Kinase ◽  
High Density ◽  
Human Neutrophils ◽  
Cross Linking ◽  
Src Kinases ◽  
Membrane Domains ◽  
Detergent Resistant Membranes

We have previously shown that CD32A (or FcγRIIA), one of the main opsonin receptors, was rapidly insolubilized and degraded in intact neutrophils after its cross-linking. In view of these experimental difficulties, the early signalling steps in response to CD32A activation were studied in purified plasma membranes of neutrophils. After CD32A cross-linking in these fractions, the tyrosine phosphorylation of two major substrates, the receptor itself and the tyrosine kinase Syk, was observed. Phosphorylation of these two proteins was observed only in the presence of orthovanadate, indicating the presence, in the membranes, of one or more tyrosine phosphatases that maintain CD32A dephosphorylation. The tyrosine phosphorylation of these two proteins was inhibited by the Src kinase inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2). The ligation of CD32A led to its recruitment to a previously uncharacterized subset of high-density flotillin-1-positive DRMs (detergent-resistant membranes). The changes in the solubility properties of CD32A were observed in the absence of added ATP; therefore, they were probably not secondary to the tyrosine phosphorylation of the receptor, rather they preceded it. Src kinases as well as Syk were constitutively present in DRMs of high and low density and no evident changes in their distribution were detected after cross-linking of CD32A. Pretreatment of plasma membranes with methyl-β-cyclodextrin did not inhibit the recruitment of CD32A to DRMs, although it led to the loss of the Src kinase Lyn from these fractions. In addition, methyl-β-cyclodextrin inhibited the tyrosine phosphorylation of CD32A and Syk induced by cross-linking of CD32A. This membrane model allowed us to observe a movement of CD32A from detergent-soluble regions of the membranes to DRMs, where it joined Src kinases and Syk and became tyrosine-phosphorylated.

Germin: physicochemical properties of the glycoprotein which signals onset of growth in the germinating wheat embryo

1987 ◽  
Vol 65 (12) ◽  
pp. 1039-1048 ◽  
Author(s):  
William C. McCubbin ◽  
Cyril M. Kay ◽  
Theresa D. Kennedy ◽  
Byron G. Lane
Keyword(s):  
Circular Dichroism ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sedimentation Coefficient ◽  
Helical Structure ◽  
Random Coil ◽  
Sedimentation Equilibrium ◽  
Cross Linking ◽  
Wheat Embryo ◽  
Circular Dichroïsm

The size and structure of germin, the homooligomeric glycoprotein which marks the onset of growth in germinating wheat embryos, has been examined by gel filtration, ultracentrifugation, electron microscopy, chemical cross-linking, and optical techniques (circular dichroism). Germin has a sedimentation coefficient (S20,w) of 7.3S, and a Stokes' radius (RS) of 4.5 nm, the latter value being compatible with the dimensions of the particle observed by negative staining in the electron microscope. By three methods (sedimentation equilibrium, sodium dodecyl sulphate (SDS) – polyacrylamide electrophoresis, S20,w/RS), the mean particle mass of the two closely related forms of germin (G and G′) is ca. 130 kilodaltons (kDa). Cross-linking with dimethyl suberimidate indicates that the oligomer is homopentameric, compatible with the molecular mass of the protomer (ca. 26 kDa) as determined by SDS–polyacrylamide gel electrophoresis. Using the Provencher and Glockner analysis to interpret circular dichroism measurements (in the far ultraviolet), both forms of germin contain about 10–20% α-helical structure, 50–60% β-sheet/turn structure, and 20–30% random coil. In a structure-inducing environment (45% trifluoroethanol), the α-helical structure increases to a value (35–40%) similar to that predicted by Chou–Fasman analysis of the protein sequence deduced by cDNA sequencing.

Tyrosine phosphorylation of specific proteins after mitogen stimulation of chicken embryo fibroblasts

1983 ◽  
Vol 3 (3) ◽  
pp. 380-390
Author(s):  
K D Nakamura ◽  
R Martinez ◽  
M J Weber
Keyword(s):  
Growth Factor ◽  
Tyrosine Phosphorylation ◽  
Chicken Embryo ◽  
Sodium Dodecyl ◽  
Biological Response ◽  
Egf Receptors ◽  
Phosphorylation Of Proteins ◽  
Embryo Fibroblasts ◽  
Chicken Embryo Fibroblasts ◽  
Stimulation Of

We found that stimulation of density-inhibited chicken embryo fibroblasts with serum, epidermal growth factor (EGF), platelet-derived growth factor, (PDGF), or multiplication-stimulating activity (MSA) leads to an increase in tyrosine phosphorylation of proteins in the region of Mr 40,000 (40K) to 42K. The increase in tyrosine phosphorylation after serum or EGF stimulation was transient, reaching a maximum at about 5 min and then declining. By fine-resolution analysis of proteins separated on sodium dodecyl sulfate-polyacrylamide gels, we found that after EGF stimulation, the major increase in phosphotyrosine content was in a 42K Mr protein, with a smaller increase in a 40K Mr protein. The increased phosphorylation in the 40K to 42K Mr region accounted for almost all of the increase in phosphotyrosine observed in these cells. These phosphotyrosine-containing proteins were different from the major phosphotyrosine-containing protein of Rous sarcoma virus-transformed chicken embryo fibroblasts, which migrates at an approximate Mr of 36K. Increased tyrosine phosphorylation of proteins of similar Mr was found in 3T3 cells treated with EGF, but not in NR-6 cells, which lack detectable EGF receptors. It is possible that the 40K to 42K Mr phosphotyrosine-containing proteins are involved in the integration of the biological response to a number of different growth factors.

Stimulation of tyrosine phosphorylation of distinct proteins in response to antibody-mediated ligation and clustering of alpha 3 and alpha 6 integrins

1995 ◽  
Vol 108 (3) ◽  
pp. 1165-1174
Author(s):  
K. Jewell ◽  
C. Kapron-Bras ◽  
P. Jeevaratnam ◽  
S. Dedhar
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Kinase Inhibitor ◽  
Cell Attachment ◽  
Umbilical Vein ◽  
Integrin Receptors ◽  
Human Prostate Carcinoma ◽  
Beta 1 Integrin ◽  
Umbilical Vein Endothelial Cells ◽  
Stimulation Of

The interaction of cells with components of the extracellular matrix through their integrin receptors results in the stimulation of tyrosine phosphorylation of several proteins, suggesting that these receptors play a key role in signal transduction. Here we report that antibody-mediated ligation and clustering of alpha 3 beta 1 and alpha 6 beta 1/alpha 6 beta 4 integrins resulted in the stimulation of tyrosine phosphorylation of proteins that are specific for each heterodimer. Thus, ligation and clustering of the alpha 3 beta 1 integrin on human prostate carcinoma cells (PC-3) and human umbilical vein endothelial cells (HUVEC) with anti-alpha 3 antibodies resulted in the stimulation of tyrosine phosphorylation of a 55 kDa protein. In contrast, ligation and clustering of the alpha 6 beta 1 integrin on these cells with anti-alpha 6 antibody resulted in the dramatic stimulation of tyrosine phosphorylation of a 90 kDa protein in addition to a 52 kDa protein, and ligation and clustering of alpha 5 beta 1 on HUVEC did not result in the apparent stimulation of tyrosine phosphorylation of any proteins. Clustering with anti-beta 1 antibodies triggered the tyrosine phosphorylation of all of these proteins, whereas ligation and clustering of PC-3 cells with an anti-beta 4 antibody resulted in the tyrosine phosphorylation of a distinct 62 kDa protein. Since the PC-3 cells express both alpha 6 beta 1 and alpha 6 beta 4, these data suggest that these two receptors can transduce distinct signals. All of the phosphorylations could be inhibited by treating the cells with Genistein, a tyrosine kinase inhibitor. Antibody-mediated ligation and clustering of integrins on the two types of cells did not result in the stimulation of tyrosine phosphorylation of pp125 focal adhesion kinase, although this was observed upon cell attachment and spreading on fibronectin, laminin and anti-alpha 3 monoclonal antibody. Collectively, these data demonstrate that cross-linking of different integrin heterodimers can stimulate tyrosine kinase activities, leading to the phosphorylation of distinct proteins, which are also different from those observed when cells are allowed to spread on a matrix.

scholarly journals Role of JAM-A tyrosine phosphorylation in epithelial barrier dysfunction during intestinal inflammation

2019 ◽  
Vol 30 (5) ◽  
pp. 566-578 ◽  
Author(s):  
Shuling Fan ◽  
Caroline M. Weight ◽  
Anny-Claude Luissint ◽  
Roland S. Hilgarth ◽  
Jennifer C. Brazil ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Barrier Function ◽  
Intestinal Inflammation ◽  
Kinase Inhibitor ◽  
Src Kinase ◽  
Small Gtpase ◽  
Epithelial Barrier ◽  
Barrier Dysfunction ◽  
Junction Protein

Junctional adhesion molecule-A (JAM-A), an epithelial tight junction protein, plays an important role in regulating intestinal permeability through association with a scaffold signaling complex containing ZO-2, Afadin, and the small GTPase Rap2. Under inflammatory conditions, we report that the cytoplasmic tail of JAM-A is tyrosine phosphorylated (p-Y280) in association with loss of barrier function. While barely detectable Y280 phosphorylation was observed in confluent monolayers of human intestinal epithelial cells under basal conditions, exposure to cytokines TNFα, IFNγ, IL-22, or IL-17A, resulted in compromised barrier function in parallel with increased p-Y280. Phosphorylation was Src kinase dependent, and we identified Yes-1 and PTPN13 as a major kinase and phosphatase for p-JAM-A Y280, respectively. Moreover, cytokines IL-22 or IL-17A induced increased activity of Yes-1. Furthermore, the Src kinase inhibitor PP2 rescued cytokine-induced epithelial barrier defects and inhibited phosphorylation of JAM-A Y280 in vitro. Phosphorylation of JAM-A Y280 and increased permeability correlated with reduced JAM-A association with active Rap2. Finally, we observed increased phosphorylation of Y280 in colonic epithelium of individuals with ulcerative colitis and in mice with experimentally induced colitis. These findings support a novel mechanism by which tyrosine phosphorylation of JAM-A Y280 regulates epithelial barrier function during inflammation.

scholarly journals Evidence for the participation of calmodulin in stimulus–secretion coupling in the pancreatic β-cell

1980 ◽  
Vol 192 (3) ◽  
pp. 919-927 ◽  
Author(s):  
Juan J. Gagliardino ◽  
Donna E. Harrison ◽  
Michael R. Christie ◽  
Elma E. Gagliardino ◽  
Stephen J. H. Ashcroft
Keyword(s):  
Insulin Release ◽  
Kinase Inhibitor ◽  
Potent Inhibitor ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Β Cell ◽  
Batch Type ◽  
Dependent Inhibition ◽  
Stimulus Secretion Coupling ◽  
Rat Islets Of Langerhans

1. The ability of a range of phenothiazines to inhibit activation of brain phosphodiesterase by purified calmodulin was studied. Trifluoperazine, prochlorperazine and 8-hydroxyprochlorperazine produced equipotent dose-dependent inhibition with half-maximum inhibition at 12μm. When tested at 10 or 50μm, 7-hydroxyprochlorperazine was a similarly potent inhibitor. However, trifluoperazine-5-oxide and N-methyl-2-(trifluoromethyl)phenothiazine were ineffective at concentrations up to 50μm, and produced only a modest inhibition at 100μm. 2. The same phenothiazines were tested for their ability to inhibit activation of brain phosphodiesterase by boiled extracts of rat islets of Langerhans. At a concentration of 20μm, 70–80% inhibition was observed with trifluoperazine, prochlorperazine, 7-hydroxyprochlorperazine or 8-hydroxyprochlorperazine, whereas trifluoperazine-5-oxide and N-methyl-2-(trifluoromethyl)phenothiazine were less effective. 3. The effect of these phenothiazines on insulin release from pancreatic islets was studied in batch-type incubations. Insulin release stimulated by glucose (20mm) was markedly inhibited by 10μm-trifluoperazine or -prochlorperazine and further inhibited at a concentration of 20μm. 8-Hydroxyprochlorperazine (20μm) was also a potent inhibitor but 7-hydroxyprochlorperazine (20μm) elicited only a modest inhibition of glucose-stimulated insulin release; no inhibition was observed with trifluoperazine-5-oxide or N-methyl-2-(trifluoromethyl)phenothiazine. 4. Trifluoperazine (20μm) markedly inhibited insulin release stimulated by leucine or 4-methyl-2-oxopentanoate in the absence of glucose, and both trifluoperazine and prochlorperazine (20μm) decreased insulin release stimulated by glibenclamide in the presence of 3.3mm-glucose. 5. None of the phenothiazines affected basal insulin release in the presence of 2mm-glucose. 6. Trifluoperazine (20μm) did not inhibit islet glucose utilization nor the incorporation of [3H]leucine into (pro)insulin or total islet protein. 7. Islet extracts catalysed the incorporation of 32P from [γ-32P]ATP into endogenous protein substrates. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis resolved several phosphorylated bands, but incorporation was slight. However, calmodulin in the presence of Ca2+ greatly enhanced incorporation: the predominant phosphorylated band had an estimated mol.wt. of 55000. This enhanced incorporation was abolished by trifluoperazine, but not by cyclic AMP-dependent protein kinase inhibitor protein. 8. These results suggest that islet phosphodiesterase-stimulating activity is similar to, although not necessarily identical with, calmodulin from skeletal muscle; that islet calmodulin may play an important role in Ca2+-dependent stimulus–secretion coupling in the β-cell; and that calmodulin may exert part at least of its effect on secretion via phosphorylation of endogenous islet proteins.

Substitution of the γ-chain Asn308 disturbs the D:D interface affecting fibrin polymerization, fibrinopeptide B release, and FXIIIa-catalyzed cross-linking

Blood ◽  
2004 ◽  
Vol 103 (11) ◽  
pp. 4157-4163 ◽  
Author(s):  
Nobuo Okumura ◽  
Oleg V. Gorkun ◽  
Fumiko Terasawa ◽  
Susan T. Lord
Keyword(s):  
High Performance ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Factor Xiiia ◽  
Cross Linking ◽  
Dimer Formation ◽  
Polymer Formation ◽  
Crystallographic Structures ◽  
Link Formation

Abstract Crystallographic structures indicate that γ-chain residue Asn308 participates in D:D interactions and indeed substitutions of γAsn308 with lysine or isoleucine have been identified in dysfibrinogens with impaired polymerization. To probe the role of Asn308 in polymerization, we synthesized 3 variant fibrinogens: γAsn308 changed to lysine (γN308K), isoleucine (γN308I), and alanine (γN308A). We measured thrombin-catalyzed polymerization by turbidity, fibrinopeptide release by high-performance liquid chromatography, and factor XIIIa–catalyzed cross-linking by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. In the absence of added calcium, polymerization was clearly impaired with all 3 variants. In contrast, at 0.1 mM calcium, only polymerization of γN308K remained markedly abnormal. The release of thrombin-catalyzed fibrinopeptide B (FpB) was delayed in the absence of calcium, whereas at 1 mM calcium FpB release was delayed only with γN308K. Factor XIIIa–catalyzed γ-γ dimer formation was delayed with fibrinogen (in absence of thrombin), whereas with fibrin (in presence of thrombin) γ-γ dimer formation of only γN308K was delayed. These data corroborate the recognized link between FpB release and polymerization. They show fibrin cross-link formation likely depends on the structure of protofibrils. Together, our results show substitution of Asn308 with a hydrophobic residue altered neither polymer formation nor polymer structure at physiologic calcium concentrations, whereas substitution with lysine altered both.

Sign in / Sign up

Export Citation Format

Share Document