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Biomedical subjects

J C Holt

Publications and source records attributed to J C Holt.

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

Functional effects of LC1-reassociation with cardiac papain Mg.S1.

The effect of LC1 on cardiac myosin structure and activity was investigated using as a model S1 prepared by papain digestion in the presence of Mg2+. The resulting S1 contained LC2 but a part of the N-terminal region of LC1 was cleaved. Sequencing the N-terminal part of the band migrating below LC1 on SDS gels revealed it to consist of alternating alanyl and prolyl residues thus establishing LC1 as the origin of this band. However, Western blots did not reveal any LC1 while radioimmunoassays indicated it to be present at the 5% level suggesting the anti-LC1 antibody used in these experiments did not recognize the C-terminal portion of LC1 still attached to Mg.S1. Mixing a 10-15 M excess of isolated light chains with Mg.S1 in the presence of 10 mM ATP, 12 mM MgCl2, 4.7 M NH4Cl allowed LC1 to recombine with LC1-deficient Mg.S1. Equilibrium ultracentrifugation analysis revealed a highly heterogeneous LC1-deficient S1 which upon recombination with intact LC1 became monodisperse as indicated by the superimposition of molecular weight averages all across the centrifuge cell. LC1-deficient Mg.S1 had a Vm of 0.4 s-1, Ka of 30 microM and a Kbind of 28 microM. In the presence of intact LC1, Vm rose to 0.8 s-1 while Ka and Kbind were reduced to 7.5 and 12 microM, respectively. The fourfold decrease in Ka strongly indicated an increased affinity for actin by Mg.S1 in the presence of uncleaved LC1. Also, Ca(2+)-regulation of dog heart myofibrils was suppressed when Ca(2+)-activated MgATPase assays, as a function of Ca2+, were performed in the presence of anti-LC1 antibodies. These observations suggest the presence of intact, uncleaved LC1 in S1 is required for the stability of S1 heavy chains and proper Ca(2+)-regulation.

Actins↗
PubMed1993

Neutrophil-activating intercrine secreted by porcine platelets is active without proteolytic processing.

A new member of the cytokine intercrine alpha-subfamily, porcine neutrophil-activating peptide 2 (pNAP-2), was isolated to homogeneity. Amino acid sequencing analysis showed two species of pNAP-2, a long form (pNAP-2-L) and a short form (pNAP-2-S). pNAP-2-L had seven more amino acids at the NH2-terminus than pNAP-2-S. The remaining amino acid sequences of the two molecules were identical. pNAP-2-S shared 65% homology with human neutrophil-activating peptide 2 (hNAP-2) including four cysteines in identical positions. Moreover, the NH2-terminal sequence Glu-Leu-Arg (E-L-R) was conserved in both molecules. Both pNAP-2-L and pNAP-2-S induced mobilization of cytosolic calcium in neutrophils and caused release of granulocyte elastase in a dose-dependent manner, although pNAP-2-L was less active. A desensitization study suggested that both hNAP-2 and pNAP-2-S may act on the same receptor. Whereas human platelets release inactive precursors that can be converted to hNAP-2 by cathepsin G from activated neutrophils, porcine platelets, upon stimulation with thrombin, appear to secrete active forms of pNAP-2. The activated neutrophils are not involved in the generation of pNAP-2.

Amino Acid Sequence↗
PubMed1992

Isolation, characterization, and immunological detection of neutrophil-activating peptide 2: a proteolytic degradation product of platelet basic protein.

Neutrophil-activating peptide 2 (NAP-2), corresponding to platelet basic protein fragment 25-94, was prepared by chymotryptic digestion of its precursors, low affinity platelet factor 4 or beta-thromboglobulin, followed by purification by high performance liquid chromatography. NAP-2 (0.1-1.5 microns) caused the release of human granulocyte elastase from cytochalasin B-treated neutrophils in a dose-dependent manner. In the same system, beta-thromboglobulin, human platelet factor 4, S-pyridylethyl NAP-2, and platelet basic protein C-terminal fragment (77-94) were inactive, whereas platelet basic protein fragment 22-89 had low, but significant, activity. Sensitive immunological identification of NAP-2 based on nonequilibrium isoelectric focusing and immunoblotting is described.

Amino Acid Sequence↗
PubMed1991

Ovine platelet factor 4: purification, amino acid sequence, radioimmunoassay and comparison with platelet factor 4 of other species.

A simple method of purification of ovine platelet factor 4 (PF4) is described. Material released by freezing and thawing suspension of washed sheep platelets was fractionated by heparin-agarose chromatography and reverse phase HPLC. Purified ovine PF4 contained 85 amino acids (Mr 9130) and showed 78% homology with bovine PF4, 76% with porcine PF4, 71% homology with human PF4, and 61% with rat PF4. The heparin binding site of ovine PF4 localized in the C-terminal region of the molecule was identified as LYKKIIKRLL. The content of PF4 determined by radioimmunoassay was 22.6 (+/- 1.6 S.D.) micrograms per 10(9) platelets and 46.8 (+/- 19.6 S.D.) ng per ml platelet poor plasma collected in acid citrate dextrose in the presence of prostaglandin E1. PF4 was rapidly released during stimulation of ovine platelets by collagen.

Amino Acid Sequence↗
PubMed1990

Batroxostatin, an Arg-Gly-Asp-containing peptide from Bothrops atrox, is a potent inhibitor of platelet aggregation and cell interaction with fibronectin.

A potent inhibitor of platelet aggregation and cell adhesion was isolated from the venom of Bothrops atrox. This peptide, referred to as batroxostatin, was composed of 71 amino acids and showed a high degree of homology with other snake venom peptides including trigramin, albolabrin, elegantin and applagin: all 12 cysteines and the RGD sequence (standard one-letter amino acid codes) aligned in the same position. Compared on a molar basis, the anti-platelet aggregation activity of batroxostatin was about 1000-times higher than that of RGDS. In addition, batroxostatin was about 400-times more potent than GRGDS at inhibiting melanoma cell adhesion to fibronectin. Batroxostatin covalently attached to plastic promoted adhesion of melanoma cells. The anti-GP140 antibody, recognizing beta 1 integrins, completely inhibited adhesion of mouse melanoma cells to batroxostatin. This observation, in addition to the inhibitory effect of batroxostatin on the adhesion of chick fibroblasts to fibronectin, suggests that batroxostatin interacts with integrins from both the beta 1 and beta 3 subfamilies.

Amino Acid Sequence↗
PubMed1990

Human platelet factor 4 and its C-terminal peptides: heparin binding and clearance from the circulation.

Human platelet factor 4 (PF4), a high affinity heparin binding protein, is released from stimulated platelets and stored at vascular sites, predominantly in liver, from where it can be brought back into circulation by heparin. We attempted to define structural requirements for PF4 binding to heparin and for the pattern of its clearance from the circulation. Intact PF4 bound strongly to heparin agarose and was eluted at 1.4 M NaCl, while reduced PF4 and PF4 C-terminal peptides PF4 (47-70) and PF4 (58-70) bound weakly and were eluted at 0.2-0.5 M NaCl. 125I-radiolabeled intact PF4, reduced PF4 and C-terminal PF4 peptides injected into rabbits were cleared from the circulation in a biphasic pattern with components having half-life time of 1-2 min and 20-140 min. Heparin eliminated the fast component of PF4 clearance, but it did not affect clearance of reduced PF4 or C-terminal PF4 peptides. In contrast to reduced PF4 and PF4 (47-70), intact PF4 that accumulated in the liver and spleen, was displaced by heparin into circulating blood. In conclusion, specific binding sites and native conformation of the molecule are critical for high affinity PF4 binding to insolubilized heparin and for a pattern of PF4 clearance from the circulation in the presence of heparin.

Animals↗
PubMed1990

Disintegrins: a family of integrin inhibitory proteins from viper venoms.

Disintegrins represent a new class of low molecular weight, RGD-containing, cysteine-rich peptides isolated from the venom of various snakes. They interact with the beta 1 and beta 3 families of integrins and their potency is at least 500-2000 times higher than short RGDX peptides. Analysis of the amino acid sequences of 14 different disintegrins suggests that the RGD sequence, in the spatial configuration determined by the appropriate pairing of the cysteine residues, functions as a cell recognition site. However, certain nonconserved amino acids appear to modify the activity of disintegrins, their specificity for various receptors, and their ability to compete specifically with various ligands.

Amino Acid Sequence↗
PubMed1989

Structural and functional characterization of major platelet membrane components derived by limited proteolysis of glycoprotein IIIa.

The authors isolated a product of proteolytic degradation of glycoprotein IIIa (GPIIIa) which is formed on the surface of human platelets during incubation with chymotrypsin and which was previously described as the 66 kDa platelet membrane component. This component migrated with an apparent Mr 62,400 in a non-reduced system of sodium dodecyl sulfate polyacrylamide gel electrophoresis. In a reduced system it yielded two major subunits migrating with apparent Mr 14,000-17,000 and 65,000. The low-molecular weight component began with the NH2-terminal sequence of GPIIIa (GPNICTTR...) and the larger component with residue 348 of GPIIIa (GKIRSKKA...) as deduced from a cDNA clone of this glycoprotein. The two subunits appeared to be linked by one or more S-S bridges supporting the contention that GPIIIa is a highly folded molecule on the platelet membrane. In contrast to GPIIIa, the '66 kDa component' did not bind to GRGDSPK-agarose, to fibrinogen-agarose nor to insolubilized monoclonal antibody recognizing the GPIIb/IIIa complex. The exposure of fibrinogen receptors during the course of incubation of platelets with chymotrypsin preceded the formation of the '66 kDa component' characterized in this study. An intermediate product of GPIIIa proteolysis migrating with an apparent Mr 120,000 in a non-reduced system and Mr 80,000 in a reduced system was identified as a precursor of the '66 kDa component'. The '120 kDa component' was not retained on GRGDSPK-agarose or on fibrinogen-agarose but it was retained on insolubilized antibody recognizing the GPIIb/IIIa complex. Incubation of platelets with porcine pancreatic elastase or human granulocytic elastase resulted in the formation of similar proteolytic degradation fragments.

Amino Acid Sequence↗
PubMed1989

Trigramin: primary structure and its inhibition of von Willebrand factor binding to glycoprotein IIb/IIIa complex on human platelets.

Trigramin, a naturally occurring peptide purified from Trimeresurus gramineus (T. stejnegeri formosensis) snake venom, inhibits platelet aggregation and the binding of 125I-fibrinogen to ADP-stimulated platelets (Ki = 2 X 10(-8) M) without affecting the platelet-release reaction. 125I-trigramin binds to ADP-stimulated and to chymotrypsin-treated normal platelets but not to thrombasthenic platelets. 125I-trigramin binding to platelets is blocked by monoclonal antibodies directed against the glycoprotein IIb/IIIa complex and by Arg-Gly-Asp-Ser (RGDS) [Huang et al. (1987) J. Biol. Chem. 262, 161]. We determined the primary structure of trigramin, which is composed of a single polypeptide chain of 72 amino acid residues and six disulfide bridges. The molecular weight of trigramin calculated on the basis of amino acid sequence was 7500, and the average pI was 5.61. An RGD sequence appeared in the carboxy-terminal domain of trigramin. An amino-terminal fragment (7-33) of trigramin showed 39% homology with a region (1555-1581) of von Willebrand factor (vWF). Trigramin also showed 36% identity in a 42 amino acid overlap and 53% identity in a 15 amino acid overlap when compared with two adhesive proteins, collagen alpha 1 (I) and laminin B1, respectively. Trigramin blocked binding of human vWF to the glycoprotein IIb/IIIa complex in thrombin-activated platelets in a dose-dependent manner. Reduction of trigramin resulted in a marked decrease in its ability to block vWF binding to human platelets.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1988

Occurrence of platelet basic protein, a precursor of low affinity platelet factor 4 and beta-thromboglobulin, in human platelets and megakaryocytes.

beta-thromboglobulin antigen, a platelet-specific secreted protein, occurs in three forms: platelet basic protein, low affinity platelet factor 4, and beta-thromboglobulin. The combined level of beta-thromboglobulin antigen in megakaryocytes, measured by radioimmunoassay, was 13 +/- 7 micrograms/10(6) cells (SD, n = 6). The relative proportions of the three forms of beta-thromboglobulin antigen present within platelets and megakaryocytes were determined in cells lysed with trichloroacetic acid to minimize artifactual proteolysis. Samples were analyzed by isoelectric focusing in polyacrylamide gel with quantitative immunological detection on a nitrocellulose transfer of the gel. In platelets, the major species found was low affinity platelet factor 4 with precursor platelet basic protein as only 25% +/- 11% (SD, n = 16) of total beta-thromboglobulin antigen. In megakaryocytes partially purified both from normal bone marrow aspirates and from whole marrow specimens obtained after surgery, platelet basic protein was a higher proportion of beta-thromboglobulin antigen (49% +/- 13% SD, n = 11) than was the case in platelets. beta-thromboglobulin itself was never detected under the conditions of cell lysis used. Our results suggest that platelet basic protein is synthesized in megakaryocytes and that its cleavage is associated with an earlier stage of cell development than simply maturation to platelets. Further support for the precursor status of platelet basic protein was found in the expression of predominantly this antigenic form in a human erythroleukemia cell line.

Antigens, Human Platelet↗
PubMed1987

Trigramin. A low molecular weight peptide inhibiting fibrinogen interaction with platelet receptors expressed on glycoprotein IIb-IIIa complex.

Trigramin, a highly specific inhibitor of fibrinogen binding to platelet receptors, was purified to homogeneity from Trimeresurus gramineus snake venom. Trigramin is a single chain (approximately 9 kDa) cysteine-rich peptide with the Glu-Ala-Gly-Glu-Asp-Cys-Asp-Cys-Gly-Ser-Pro-Ala NH2-terminal sequence. Chymotryptic fragmentation showed the Arg-Gly-Asp sequence in trigramin. Trigramin inhibited fibrinogen-induced aggregation of platelets stimulated by ADP (IC50 = 1.3 X 10(-7)M) and aggregation of chymotrypsin-treated platelets. It did not affect the platelet secretion. Trigramin was a competitive inhibitor of the 125I-fibrinogen binding to ADP-stimulated platelets (Ki = 2 X 10(-8) M). 125I-Trigramin bound to resting platelets (Kd = 1.7 X 10(-7) M; n = 16,500), to ADP-stimulated platelets (Kd = 2.1 X 10(-8) M; n = 17,600), and to chymotrypsin-treated platelets (Kd = 8.8 X 10(-8) M; n = 13,800) in a saturable manner. The number of 125I-trigramin binding sites on thrombasthenic platelets amounted to 2.7-5.4% of control values obtained for normal platelets and correlated with the reduced number of GPIIb-GPIIIa molecules on the platelet surface. EDTA, monoclonal antibodies directed against the GPIIb-GPIIIa complex, and synthetic peptides (Arg-Gly-Asp-Ser and Tyr-Gly-Gln-Gln-His-His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val) blocked both 125I-fibrinogen binding and 125I-trigramin binding to platelets. Fibrinogen binding was more readily inhibited by these compounds than was trigramin binding. Monoclonal antibodies directed either against GPIIb or GPIIIa molecules did not block the interaction of either ligand with platelets. Reduced, S-pyridylethyl, trigramin did not inhibit platelet aggregation and fibrinogen binding to platelets and it did not bind to platelets, suggesting that the secondary structure of this molecule is critical for expression of its biological activity.

Adenosine Diphosphate↗
PubMed1986

Characterization of human platelet basic protein, a precursor form of low-affinity platelet factor 4 and beta-thromboglobulin.

Platelet basic protein (PBP) was purified from the supernatant of thrombin-stimulated, washed human platelets by ion-exchange, affinity, molecular sieve, and high-performance liquid chromatography (HPLC). The NH2-terminal amino acid sequence was determined by automated Edman degradation, revealing 9 unique residues followed by 10 residues of the established low-affinity platelet factor 4/beta-thromboglobulin (LA-PF4/beta TG) sequence. Among the nine were three basic residues, accounting for the high isoelectric point of PBP. Additional evidence for precursor status includes the immunological cross-reactivity of all three species and the ability of plasmin and trypsin to produce from PBP a species resembling beta TG in charge, hydrophobicity, and size. Tryptic peptide maps of PBP and LA-PF4 obtained by reverse-phase HPLC were very similar, and from each protein, a peptide was isolated which showed the amino acid composition predicted for the COOH-terminal tryptic peptide of beta TG. Normal platelets contained predominantly LA-PF4, with PBP ranging from 10% to 30% of total beta TG antigen. This was true even when fresh platelets were lysed with trichloroacetic acid in order to provide the most complete and rapid inhibition of proteolytic activity. beta TG itself was never detected in this situation or in the release supernatant of stimulated platelets, and only rarely in unprotected lysates. In agreement with earlier results, crude preparations of PBP were mitogenic for 3T3 cells, but highly purified preparations of PBP and LA-PF4 were free of this activity.

Amino Acid Sequence↗
PubMed1985

The interaction of human platelet thrombospondin with fibrinogen. Thrombospondin purification and specificity of interaction.

Human platelet thrombospondin (TSP) was purified to homogeneity by chromatography on fibrinogen coupled to cyanogen bromide-activated Sepharose. The yield of TSP was 1.3 mg or approximately 22% of that present in platelet-rich plasma as determined by radioimmunoassay. It analyzed on discontinuous sodium dodecyl sulfate gels as a single band having apparent molecular weights of 180,000 and greater than 400,000 under reducing and nonreducing conditions, respectively. Amino acid analysis gave results similar to previously published values. Antibodies raised in rabbits were monospecific as evaluated by radioimmunoassay. In double immunodiffusion tests, these antibodies gave one line of identity against TSP purified by this procedure and TSP purified by published procedures, confirming the identity of the material isolated. The protein possesses no lectin-like activity. The specificity of the TSP-fibrinogen interaction was investigated. TSP binding to fibrinogen-Sepharose occurred in the presence of EDTA, indicating that calcium and magnesium ions are not required for interaction of TSP with fibrinogen. The binding of TSP to fibrinogen-Sepharose was quantitatively blocked by pretreatment with an antibody to the cyanogen bromide cleavage fragment composed of residues 241-476 of the carboxyl-terminal end of the alpha chain of fibrinogen. Antibodies against the D and E domains of fibrinogen had no effect on the binding. Excess fibrinogen (30 mg/ml) added to platelet extract quantitatively inhibited binding of TSP to fibrinogen-Sepharose. TSP preferentially bound to uncross-linked fibrin, suggesting that the TSP-fibrinogen binding site is unavailable in cross-linked fibrin. These results indicate that TSP binds specifically to immobilized fibrinogen or uncross-linked fibrin through determinants present in the carboxyl-terminal portion of the alpha chain and that these interactions do not require calcium or magnesium ions.

Amino Acids↗
PubMed1985

Identification of PlAl alloantigen domain on a 66 kDa protein derived from glycoprotein IIIa of human platelets.

Incubation of platelets with chymotryptin leads to the exposure of fibrinogen receptors and to the appearance of a 66 kDa membrane component on the surface of platelets. Both glycoprotein IIIa (GP IIIa) and a 66 kDa component were precipitated from detergent extracts of solubilized, surface radiolabeled chymotrypsin-treated platelets by human anti-PlAl antisera. Moreover, the presence of the P1A1 antigen was identified on GP IIIa (but not on GP IIb) and on a 66 kDa protein by means of immunoblot procedures using platelet Triton X-114 extracts and these purified proteins. Anti-PlAl antiserum did not recognize GP IIIa on the surface of intact (untreated) platelets nor the 66 kDa protein on the surface of chymotrypsin-treated platelets of PlAl-negative individuals. The present data demonstrate directly that the 66 kDa protein is derived from GP IIIa and contains the PlAl alloantigen.

Antigens, Human Platelet↗
PubMed1984

Partial purification and characterization of porcine platelet-derived growth factor (PDGF).

Platelet derived growth factor (PDGF) has been partially purified from porcine platelets. Purification steps included heparin-agarose chromatography of the material released by thrombin-stimulated washed porcine platelets and Blue-Sepharose chromatography. Preparative isoelectric focusing showed that isoelectric point of porcine PDGF is at pH 10.0-11.0 and elution experiments from sodium dodecyl sulfate (SDS) polyacrylamide gels indicated that its molecular weight is close to 30 kD. The immunoglobulin fraction prepared from anti-human PDGF serum inhibited the mitogenic activity of porcine PDGF. These experiments suggest a homology of porcine and human PDGF. Porcine platelet factor 4 and porcine platelet basic protein were devoid of significant mitogenic activity.

Animals↗
PubMed1983

Purification of two heparin-binding proteins from porcine platelets and their homology with human secreted platelet proteins.

Two heparin-neutralizing proteins secreted by thrombin-stimulated platelets were purified to homogeneity by means of heparin-agarose affinity chromatography. These proteins, termed porcine platelet basic protein (PBP) and porcine platelet factor 4 (PF4), were eluted from a heparin-agarose column at 0.6-0.9 M NaCl and at 1-1.4 M NaCl, respectively. The molecular weight of porcine platelet basic protein was 7,000-7,700 daltons, as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and amino acid analysis. The isoelectric point of this protein was at pH 9.0. The amino acid composition of porcine platelet basic protein resembled that of human low affinity platelet factor 4 (LA-PF4), except that the porcine protein did not contain tyrosine. The molecular weight of porcine platelet factor 4 ranged from 10,000 (estimated from amino acid analysis) to 14,000 (estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis). The amino acid compositions of human platelet factor 4 and of porcine platelet factor 4 were similar. Monospecific antibodies against porcine platelet factor 4 and porcine platelet basic protein were raised in rabbits. Competitive radioimmunoassay demonstrated a low but significant immunologic cross-reactivity between human and porcine platelet factor 4, and between porcine platelet basic protein and a group of human secreted platelet proteins that bind to heparin with low affinity (beta-thromboglobulin [beta TG] and low affinity platelet factor 4). Experiments with direct immuno-precipitation of 125I-labeled antigens suggested that all four proteins investigated (human platelet factor 4, porcine platelet factor 4, human low affinity platelet factor 4 or human beta-thromboglobulin, and porcine platelet basic protein) share common antigenic determinants. However, there was a higher degree of immunologic cross-reactivity between heterologous antigens with similar heparin binding affinity (human platelet factor 4 and porcine platelet factor 4) than between heterologous antigens with different binding affinity (human platelet factor 4 and porcine platelet basic protein). In conclusion, our finding suggests a significant structural homology among the four proteins.

Animals↗