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Biomedical subjects

R I Handin

Publications and source records attributed to R I Handin.

At least 37 records · Page 2Linked to original sources
PubMed1990

Transforming growth factor beta inhibits endomitosis in the Dami human megakaryocytic cell line.

Megakaryocyte development is a carefully controlled process that is at least partially regulated by cytokines. Previous investigations of megakaryocyte development have focused primarily on defining growth factors that induce or enhance differentiation. In this study we demonstrate that a specific cytokine, transforming growth factor beta 1 (TGF beta 1), inhibits the phorbol myristate acetate (PMA)-induced differentiation of the Dami human megakaryocytic cell line. The addition of purified platelet TGF beta 1 inhibits PMA-induced endomitosis in a dose-dependent manner. Inhibition of endomitosis occurs with as little as 0.4 pmol/L TGF beta 1, is half-maximal at 6.4 pmol/L, and is maximal between 40 and 200 pmol/L TGF beta 1. Inhibition does not require other growth factors or nonmegakaryocytic cells. Removal of TGF beta 1 from the cultures decreases inhibition, suggesting that the continuous presence of TGF beta 1 is required and that its effects are reversible. This effect occurs even though the Dami cells constitutively express TGF beta 1 messenger RNA (mRNA) and the TGF beta 1 mRNA levels are increased by PMA. TGF beta 1 also has been shown to inhibit endomitosis during short-term culture of primary human megakaryocytes. These results suggest a model in which negative as well as positive regulatory factors modulate a critical stage of megakaryocyte development.

Cell Differentiation↗
PubMed1990

Evidence that an abnormality in the glycoprotein Ib alpha gene is not the cause of abnormal platelet function in a family with classic Bernard-Soulier disease.

The underlying molecular basis for Bernard-Soulier Disease (BSD) is currently unknown. Platelets from patients with this autosomal recessive bleeding disorder have multiple abnormalities, including a markedly reduced von Willebrand factor-dependent adhesiveness due to a deficiency of the platelet membrane glycoprotein (GP) Ib/IX complex. In the present studies, we have used an intragenic restriction fragment length polymorphism (RFLP) for Taq I in the GPIb alpha gene to study linkage between this gene and the inheritance of BSD in a family with two affected siblings. Whereas the proband was heterozygous, showing both the 0.7 and 4.0 kb bands of this polymorphism (A/B), her affected brother was homozygous for the 0.7 kb band (A/A). Accordingly, these siblings did not inherit the same pair of GPIb alpha alleles from their parents. Additionally, one child of the proband was A/A, while the second studied child was A/B, with neither showing any evidence of BSD. No construct of heterozygosity or homozygosity for GPIb alpha alleles in this family is consistent with a model in which one or more defective GPIb alpha alleles could produce BSD. RFLP analysis with BamHI or HindIII showed entirely normal patterns in the patients, indicating the absence of any gross deletion of the GPIb alpha gene. GPIb alpha mRNA from patient platelets was reverse transcribed and subsequently amplified by the polymerase chain reaction, demonstrating the presence of GPIb alpha transcript. Furthermore, trace amounts of GPIb could be shown on the surface of patient platelets. Based on these results, a defect in the GPIb alpha gene is unlikely to be the cause of BSD in this family.

Base Sequence↗
PubMed1990

Molecular studies of von Willebrand disease: reduced von Willebrand factor biosynthesis, storage, and release in endothelial cells derived from patients with type I von Willebrand disease.

Endothelial cells were cultured from the umbilical veins of two neonates with type I von Willebrand disease (vWD) and compared with cells cultured in parallel from normal control umbilical veins. In both cases, cultured vWD endothelial cells contained less messenger RNA (mRNA) encoding von Willebrand factor (vWF), and constitutively secreted two- to fourfold less vWF protein than their matched controls. Regulated secretion of stored vWF induced by thrombin or phorbol-12-myristate-13-acetate (PMA) was also diminished in vWD cells. Both the mRNA and protein produced by each of these type I vWD cells appeared to be of normal size. However, despite the diminished size of the vWF storage pool, electron microscopy of endothelial cells in situ showed normal appearing vWF storage organelles (Weibel-Palade bodies). These studies show that cultured umbilical vein endothelial cells can be used to explore the molecular defects in type I and perhaps other forms of vWD, and suggest that at least some forms of type I vWD are caused by diminished mRNA transcription or subsequent translation due to a defective vWF allele.

Adult↗
PubMed1989

Interaction of platelet factor four with cultured vascular endothelial cells.

Platelets secrete a low-molecular-weight protein, platelet factor four (PF-4), which binds to and neutralizes heparin and related sulfated glycosaminoglycans (GAGs). To examine the interactions of PF-4 with the GAGs present on endothelial cell surfaces, we incubated 125I-PF-4 with cell suspensions derived from confluent monolayers of cultured bovine aortic endothelium. Binding of 125I-PF-4 was inhibited by a 100-fold excess of nonradioactive PF-4 and varied with duration and temperature of incubation. At 4 degrees C, binding reached equilibrium at 20 minutes with kd = 2.87 mumol/L and Bmax of 63.83 pmol/10(5) cells. Binding capacity was reduced 83.4% by brief incubation of endothelial cells with trypsin and 46.67% by incubation with Flavobacterium heparinase, but was unchanged by chondroitin-ABCase treatment. At 37 degrees C, PF-4 was internalized by confluent monolayer of bovine aortic endothelial cells primarily through low-affinity adsorptive endocytosis. The internalized PF-4 was degraded to amino acids and small peptides with 50% conversion after 18-hour incubation. These studies demonstrate that a secreted platelet protein can bind to and enter endothelial cells. Binding may explain the rapid clearance of released PF-4 from plasma and could have important local effects on endothelial structure and function.

Animals↗
PubMed1988

The propeptide of von Willebrand factor independently mediates the assembly of von Willebrand multimers.

The biosynthesis of von Willebrand Factor (vWF) by vascular endothelial cells involves a complex series of processing steps that includes proteolytic cleavage of a 741-residue propeptide and the assembly of disulfide-linked multimers. Using a model system in which experimentally altered vWF cDNAs are expressed in COS-1 cells, we have shown that the vWF propeptide contains determinants that govern the assembly of vWF multimers. Furthermore, the role of the propeptide (in the assembly process) does not require it to be a contiguous part of the pro-vWF primary structure, since independently expressed propeptide was shown to promote the assembly of mature vWF subunits into multimers. Pulse-chase experiments indicated that the independently expressed propeptide formed a transient association with the mature vWF subunit inside the cell. Thus, it appears that the vWF propeptide segment can act in "trans" to direct the assembly of disulfide-linked vWF multimers.

Animals↗
PubMed1988

Platelet storage results in a redistribution of glycoprotein Ib molecules. Evidence for a large intraplatelet pool of glycoprotein Ib.

Platelet membrane glycoprotein (GP) Ib contains receptor for von Willebrand factor and thrombin. Its proteolytic fragment, glycocalicin, circulates in normal plasma. In this study, storage of platelet concentrates for 5 d resulted in a 221% increase in plasma glycocalicin (1.3 times the total amount of glycocalicin present on the surface of all platelets), an 8% overall increase in platelet surface GPIb, and the appearance of a surface GPIb-negative subpopulation of platelets. Total platelet GPIb content of fresh washed platelets, determined by gel electrophoresis and immunoassay of Triton X-100 lysates, averaged 159,740 molecules per platelet. There were 36,360 surface GPIb molecules per platelet, determined by immunoassay of the supernatant of fresh washed platelets whose surface GPIb had been completely plasmin-cleaved. In summary, these studies provide evidence for (a) a redistribution of GPIb molecules with platelet storage, and (b) a large intraplatelet pool of GPIb (approximately threefold larger than the platelet surface pool of GPIb).

Antibodies, Monoclonal↗
PubMed1988

Characterization of a new megakaryocytic cell line: the Dami cell.

A new human megakaryocytic cell line (Dami) has been established from the blood of a patient with megakaryoblastic leukemia. The Dami cells grow primarily in suspension with a doubling time of 24 to 30 hours. By light and electron microscopy, the Dami cells range in size from 12 to 120 micron in diameter and have lobulated nuclei characteristic of megakaryocytes. At least 89% of the cells react with monoclonal antibodies against platelet glycoproteins (GP) Ib and IIB/IIIa, and glycophorin. The cells do not react with antibodies against lymphoid, monocyte, granulocyte, or macrophage antigens. Thirteen percent of the cells become polyploid, spontaneously achieving greater than 4N DNA ploidy levels. In response to phorbol myristate acetate (PMA), the proportion of cells with ploidy levels greater than 4N increased threefold and could be separated into discrete ploidy groups. PMA also increased the expression of GPIb, the GPIIb/GPIIIa complex,l and von Willebrand factor. Cytogenetic analysis revealed a human male hyperdiploid karyotype with a modal chromosome number of 54 to 64 and several consistent clonal chromosomal abnormalities. These included a partial deletion of chromosome 5 and a translocation involving chromosome 3. In contrast to other megakaryocytic cell lines in which only a small portion of the cells express the megakaryocytic phenotype, nearly all of the Dami cells express platelet glycoproteins. Thus, the Dami cells provide a superior model in which to study human megakaryocyte biochemistry and differentiation.

Antigens, Differentiation↗
PubMed1987

Composition of the von Willebrand factor storage organelle (Weibel-Palade body) isolated from cultured human umbilical vein endothelial cells.

von Willebrand factor (VWF) is a large, adhesive glycoprotein that is biosynthesized and secreted by cultured endothelial cells (EC). Although these cells constitutively release VWF, they also contain a storage pool of this protein that can be rapidly mobilized. In this study, a dense organelle fraction was isolated from cultured umbilical vein endothelial cells by centrifugation on a self-generated Percoll gradient. Stimulation of EC by 4-phorbol 12-myristate 13-acetate (PMA) resulted in the disappearance of this organelle fraction and the synchronous loss of Weibel-Palade bodies as judged by immunoelectron microscopy. Electrophoretic and serologic analyses of biosynthetically labeled dense organelle fraction revealed that it is comprised almost exclusively of VWF and its cleaved pro sequence. These two polypeptides were similarly localized exclusively to Weibel-Palade bodies by ultrastructural immunocytochemistry. The identity of the dense organelle as the Weibel-Palade body was further established by direct morphological examination of the dense organelle fraction. The VWF derived from this organelle is distributed among unusually high molecular weight multimers composed of fully processed monomeric subunits and is rapidly and quantitatively secreted in unmodified form after PMA stimulation. These studies: establish that the Weibel-Palade body is the endothelial-specific storage organelle for regulated VWF secretion; demonstrate that in cultured EC, the VWF concentrated in secretory organelles is of unusually high molecular weight and that this material may be rapidly mobilized in unmodified form; imply that proteolytic processing of VWF involved in regulated secretion takes place after translocation to the secretory organelle; provide a basis for further studies of intracellular protein trafficking in EC.

Cell Fractionation↗
PubMed1986

Propolypeptide of von Willebrand factor circulates in blood and is identical to von Willebrand antigen II.

The generally mild bleeding disorder of von Willebrand disease is associated with abnormalities of two distinct plasma proteins, the large multimeric von Willebrand factor (vWF), which mediates platelet adhesion, and von Willebrand antigen II (vW AgII), which is of unknown function. The two proteins were found to have a common biosynthetic origin in endothelial cells and megakaryocytes, which explains their simultaneous absence in the severe form of this hereditary disease. Shared amino acid sequences from a 100-kilodalton plasma glycoprotein and from vW AgII are identical to amino acid sequences predicted from a complementary DNA clone encoding the 5' end of vWF. In addition, these proteins have identical molecular weights and immunologic cross reactivities. Monoclonal antibodies prepared against both proteins recognize epitopes on the pro-vWF subunit and on a 100-kilodalton protein that are not present on the mature vWF subunit in endothelial cell lysates. In contrast, polyclonal antibodies against vWF recognize both pro-vWF and vWF subunits. Thus, the 100-kilodalton plasma glycoprotein and vW AgII are identical proteins and represent an extremely large propolypeptide that is first cleaved from pro-vWF during intracellular processing and then released into plasma.

Amino Acid Sequence↗
PubMed1986

Structural basis of von Willebrand factor binding to platelet glycoprotein Ib and collagen. Effects of disulfide reduction and limited proteolysis of polymeric von Willebrand factor.

von Willebrand factor (vWF) is a large, multimeric glycoprotein that helps platelets adhere to vascular subendothelium. Although vWF binding to platelet receptors and connective tissue constituents is of fundamental importance in adhesion, there is little information regarding the nature of these vWF binding sites. In this paper, we have compared the structural requirements for vWF binding with platelet glycoprotein Ib (GpIb), heparin, and collagen and have shown that fragments derived from large vWF multimers retain biologic activity. We have shown that a 440,000-D subunit produced by disulfide reduction and alkylation of vWF polymer binds to platelet GpIb. When analyzed by polyacrylamide gel electrophoresis and Sepharose CL6B chromatography, the 440,000-D vWF oligomer is a dimer of the 220,000 subunit of fully reduced native vWF. This vWF dimer competes with 125I-vWF for binding to GpIb with an IC50 of 100 micrograms/ml (227 nM). The GpIb binding domain on vWF was further localized by digestion of native vWF polymers with Staphylococcal V8 protease. A 285,000-D fragment of vWF multimer was separated from heterogeneous 210,000-225,000-D fragments by its ability to bind to heparin. The 285,000-D fragment that bound to heparin Sepharose was composed of two disulfide-linked 175,000- and 115,000-D polypeptides. The heterogeneous fragments contained disulfide-linked 96,000, 66,000, and 53,000-D polypeptides when analyzed on polyacrylamide gel electrophoresis. The 285,000-D fragment competed with 125I-vWF for binding to GpIb with an IC50 of 22 micrograms/ml (77 nM), while the other fragments did not compete for binding. Neither the vWF dimer nor the proteolytic fragments competed with native 125I-vWF polymer for binding to collagen.

Alkylation↗
PubMed1986

Binding and covalent cross-linking of purified von Willebrand factor to native monomeric collagen.

We have analyzed the interaction of the adhesive glycoprotein, von Willebrand factor (vWF), with native monomeric collagen monolayers by adsorbing acid soluble Types I and III collagen derived from calf skin to polystyrene microtiter wells and incubating the wells with purified human 125I-vWF. The binding of 125I-vWF was saturable, reversible, specific, and was abolished by heat denaturation of the collagen monomers. Binding was half-maximal at 5 micrograms/ml, and, at saturation, 7.5 ng 125I-vWF were bound to each microgram of immobilized collagen. 125I-vWF did not bind to wells coated with other extracellular matrix or plasma proteins such as fibronectin, fibrinogen, gelatin, or the q subunit of the first component of complement (C1q). In addition, bound 125I-vWF could not be displaced from collagen by the addition of either fibronectin or fibrinogen. After incubation with Factor XIIIa, plasma transglutaminase, 125I-vWF bound to collagen could no longer be displaced by vWF, which suggests covalent cross-linking of vWF to collagen monomers. Factor XIIIa-dependent covalent cross-linking of vWF to collagen, but not to fibronectin or laminin, was also demonstrated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate.

Animals↗
PubMed1986

von Willebrand protein facilitates platelet incorporation in polymerizing fibrin.

von Willebrand protein was found to promote the incorporation of platelets into evolving fibrin thrombi. Using formalin-treated or fresh platelets, both the initial rate and extent of platelet incorporation into polymerizing fibrin were dependent on von Willebrand protein. von Willebrand protein was incorporated into evolving fibrin thrombi in parallel with platelets. Soluble fibrin monomer covalently linked to acrylonitrile beads (Matrex 102) bound von Willebrand protein specifically and saturably with an apparent approximate dissociation constant (KD) of 15 micrograms/ml. Glycocalicin, the water-soluble proteolytic fragment of glycoprotein Ib, bound to fibrin monomer in this system specifically and saturably, as well, with an apparent approximate KD of 5 micrograms/ml, but only in the presence of saturating concentrations of von Willebrand protein. These data demonstrate that the initial rate and extent of platelet incorporation into evolving fibrin thrombi are dependent on von Willebrand protein; von Willebrand protein serves as a link between polymerizing fibrin and platelet surface glycoprotein Ib; and von Willebrand protein binds to fibrin monomer and is thereby able to bind to platelet surface glycoprotein Ib in the absence of ristocetin.

Blood Platelets↗
PubMed1986

Proteolysis of platelet glycoprotein Ib by plasmin is facilitated by plasmin lysine-binding regions.

We have characterized the effects of plasmin on glycoprotein Ib (GpIb), a platelet membrane receptor for von Willebrand factor (vWF), and on glycocalicin, a fragment of the alpha chain of GpIb that contains the vWF-binding region. The addition of 4.5 X 10(-7) mol/L plasmin to washed platelets caused a time-dependent decrease in ristocetin-induced, vWF-dependent platelet agglutination. epsilon-Aminocaproic acid (EACA) inhibited plasmin release of glycocalicin-related antigen from washed platelets and preserved vWF-dependent platelet agglutination, thus indicating that the lysine-binding sites on plasmin facilitated its degradation of GpIb. To demonstrate a direct interaction between plasmin and the vWF-binding region of GpIb we incubated purified glycocalicin with plasmin. Plasmin degraded the glycocalicin into two small carbohydrate-poor peptides and into a larger carbohydrate-rich fragment. EACA was able to inhibit plasmin-mediated degradation of glycocalicin in a concentration-dependent fashion. These studies indicated that plasmin degradation of GpIb was due to a direct interaction between plasmin and GpIb and that this effect was mediated by the lysine-binding region of the plasmin molecule.

Aminocaproates↗
PubMed1986

Partial characterization of a binding site for von Willebrand factor on glycocalicin.

The binding of von Willebrand factor (vWF) to platelet membrane glycoprotein Ib (GpIb) facilitates platelet adhesion to vascular subendothelium. In this study, we provide evidence that the vWF binding site is on glycocalicin (GC), a proteolytic fragment of GpIb, and we examine the role of the carbohydrate portion of GC on that binding. The binding to platelets of 6D1, a monoclonal antibody that recognizes an epitope on GpIb and blocks ristocetin-induced vWF binding to platelets, was inhibited by purified GC. In addition, purified GC inhibited ristocetin-dependent binding of 125I-labeled vWF to platelets. Since GC contains 60% carbohydrate by weight, we assessed the role of carbohydrate sequences on its interaction with antibody 6D1 and vWF. Based on the known sequence of the major oligosaccharide chain of GC--N-acetyl neuraminic acid, galactose, N-acetyl glucosamine, N-acetyl galactosamine--we treated GC sequentially with neuraminidase, beta-galactosidase, and beta-N-acetylglucosaminidase. Removal of sialic acid and galactose residues did not affect GC binding. Removal of N-acetyl glucosamine residues did not affect GC binding to 6D1 but did decrease the ability of GC to inhibit vWF binding to platelets, increasing the concentration needed to inhibit binding by 50% (IC50) 40-fold. This suggests that a portion of the oligosaccharide chains on GC contributes to the vWF binding activity of this molecule.

Antibodies, Monoclonal↗
PubMed1985

Solution studies of the quaternary structure and assembly of human von Willebrand factor.

The reversible association of protomers of von Willebrand protein (vWF) was studied in order to analyze the forces and mechanism of vWF polymer assembly. At concentrations of vWF found in plasma (approximately 16 micrograms/mL), disulfide bond reduction with 50 mM 2-mercaptoethanol (2-ME) markedly reduced both vWF activity, as measured by ristocetin-dependent platelet agglutination, and average polymer size (Rh, the mean hydrodynamic radius) in solution, as determined by quasi-elastic light scattering (QLS) and by gel filtration chromatography. With increasing vWF concentration, activity and Rh increased despite reduction of interprotomer disulfide bonds. Changes in temperature after 2-ME treatment produced reversible changes in activity and Rh. Varying the total vWF concentration at any given temperature after 2-ME treatment changed Rh in a consistent and predictable fashion, so that estimates of the dissociation constant for vWF protomer-polymer equilibrium were obtained: Kd5 degrees C = 0.77 micrograms/mL, Kd25 degrees C = 2.4 micrograms/mL, and Kd37 degrees C = 7.7 micrograms/mL, where under the conditions of reduction presented here, the basic protomer of vWF is a dimer. Increasing ionic strength after 2-ME treatment with 1 M KCl did not change Rh, while approximately 100 microM sodium dodecyl sulfate (SDS) or approximately 300 microM sodium deoxycholate (DOC) reduced both Rh and activity compared with those of unreduced polymer. These data show that disulfide bonds are necessary to maintain vWF polymer size and activity at plasma concentrations but that noncovalent forces of association can maintain vWF polymer size and activity at higher concentrations. These forces of association may be important for polymer assembly during intracellular synthesis of vWF.

Disulfides↗