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R K Andrews

Publications and source records attributed to R K Andrews.

45 records · Page 3Linked to original sources
PubMed1992

Identification of aspartic acid 514 through glutamic acid 542 as a glycoprotein Ib-IX complex receptor recognition sequence in von Willebrand factor. Mechanism of modulation of von Willebrand factor by ristocetin and botrocetin.

As the first step in hemostasis, the binding of von Willebrand factor (vWF) to the platelet membrane glycoprotein (GP) Ib-IX complex is essential for platelet adhesion at high-shear blood flow. This interaction in vivo requires the prior binding of vWF to the subendothelial matrix, a process which exposes a normally cryptic binding site on vWF for the GP Ib-IX complex. This process can be mimicked in vitro by modulators such as ristocetin or the snake venom protein botrocetin or by desialation of vWF. We have previously localized the GP Ib binding site on vWF to a monomeric dispase fragment which extends from Leu-480/Val-481 to Gly-718 in the primary sequence of mature vWF [Andrews, R. K., Gorman, J. J., Booth, W. J., Corino, G. L., Castaldi, P. A., & Berndt, M. C. (1989) Biochemistry 28, 8326-8336]. This fragment also contains a distinct binding site for botrocetin. Analysis of synthetic peptides corresponding to hydrophilic stretches of sequence within this fragment indicated that the sequence Asp-514-Glu-542 represents a major adhesive sequence involved in receptor recognition. This peptide inhibited both the ristocetin- and botrocetin-mediated binding of vWF to either platelets or purified GP Ib-IX complex (IC50 approximately 50-200 microM) as well as the asialo-vWF- and bovine vWF-dependent agglutination of platelets. Both the N- and C-terminal halves of the peptide were inhibitory but less so than the intact peptide. This peptide also inhibited botrocetin binding to vWF, suggesting that botrocetin modulates vWF-GP Ib interaction by binding in close proximity to the vWF adhesion sequence.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1992

Identification of a region in the cytoplasmic domain of the platelet membrane glycoprotein Ib-IX complex that binds to purified actin-binding protein.

The platelet membrane glycoprotein (GP) Ib-IX complex is a major site of attachment of the platelet membrane skeleton to the plasma membrane. This association is mediated by the interaction of actin-binding protein with the GP Ib-IX complex. The aim of the present work was to identify domains on the GP Ib-IX complex that interact with actin-binding protein. Synthetic peptides corresponding to sequences of the GP Ib alpha-chain and beta-chain cytoplasmic domains were analyzed for their ability to bind to purified actin-binding protein. Two overlapping peptides encompassing a sequence (Thr-536-Phe-568) from the central region of the cytoplasmic domain of GP Ib alpha were the most effective in binding 125I-actin-binding protein, as assessed by a microtiter well approach and peptide affinity chromatography. One of the active peptides (Thr-536-Leu-554) was chosen to evaluate the likelihood that the central region of the cytoplasmic domain of GP Ib alpha is involved in binding of the intact complex to actin-binding protein. This peptide could be specifically cross-linked to purified actin-binding protein in solution. Rabbit polyclonal antibody against this peptide inhibited the binding of purified actin-binding protein to the purified GP Ib-IX complex. Finally, as in intact platelets, the calpain-induced hydrolytic fragments of purified actin-binding protein (M(r) = 200,000 and M(r) = 91,000) showed little binding to the GP Ib alpha peptide. Taken together, these results provided evidence that a region between Thr-536 and Phe-568 of the cytoplasmic domain of GP Ib alpha participates in the interaction of the GP Ib-IX complex with actin-binding protein.

Amino Acid Sequence↗
PubMed1991

Interaction of purified actin-binding protein with the platelet membrane glycoprotein Ib-IX complex.

The interaction of platelet membrane glycoprotein (GP) Ib-IX complex with the cytoplasmic membrane skeleton is potentially of major importance in regulating platelet function. Indirect evidence suggested that this interaction is mediated by actin-binding protein, but it is not known whether GP Ib-IX and actin-binding protein associate directly. To examine more closely the nature of this association, purified GP Ib-IX complex was specifically bound and oriented on the surface of impermeable polymer beads via a monoclonal antibody, AK 2, directed against the extracytoplasmic domain of GP Ib alpha (glycocalicin). Binding was specific since 1) it was abolished by excess unlabeled actin-binding protein; 2) there was no detectable specific binding of radiolabeled actin-binding protein to beads coated with glycocalicin, the major extracytoplasmic proteolytic fragment of GP Ib alpha; and 3) unlike actin-binding protein, there was no specific binding of bovine serum albumin or human platelet vinculin to the GP Ib-IX complex-coated beads. Binding of actin-binding protein to the GP Ib-IX complex-coated beads, but not to the glycocalicin-coated beads, was saturable and reversible (apparent Kd = 1 x 10(-7) M). These experiments provide direct evidence that actin-binding protein can bind to the cytoplasmic domain of a membrane glycoprotein. Because actin-binding protein is found submembranously in cells other than the platelet, it is possible that this protein may link actin filaments to the plasma membrane in those cells.

Actins↗
PubMed1989

Characterization of human platelet GMP-140 as a heparin-binding protein.

Human platelet GMP-140 has been identified as a heparin-binding protein. Purified platelet GMP-140 bound to Heparin-Sepharose CL-6B and was eluted by approximately 0.5 M sodium chloride. Radioiodinated GMP-140 bound specifically and saturably to heparin immobilized on Matrex-Pel 102 beads. Binding of radioiodinated GMP-140 to heparin-Matrex-Pel 102 beads was divalent cation-independent and was strongly inhibited by excess fluid phase GMP-140 and heparin and by other sulfated glycans such as fucoidin and dextran-sulfate. Binding was not inhibited by chondroitins 4- and 6-sulfate or mannose 6-phosphate.

Antibodies, Monoclonal↗
PubMed1989

Purification of botrocetin from Bothrops jararaca venom. Analysis of the botrocetin-mediated interaction between von Willebrand factor and the human platelet membrane glycoprotein Ib-IX complex.

Interaction of von Willebrand factor (vWF) with its platelet receptor only occurs in vitro in the presence of a modulator such as ristocetin. We have recently confirmed that the human platelet membrane glycoprotein (GP) Ib-IX complex is the receptor involved in the ristocetin-dependent binding of vWF by reconstitution with the purified components [Berndt, M.C., Du, X., & Booth, W.J. (1988) Biochemistry 27, 633-640]. We have now developed a similar solid-phase reconstitution assay using an alternate modulator, botrocetin, for the competitive analysis of functional domains in both vWF and the GP Ib-IX complex. Botrocetin was purified from Bothrops jararaca venom by ammonium sulfate fractionation and subsequent DEAE-cellulose and hydroxylapatite chromatography. The purified protein was a 25-kilodalton (kDa) disulfide-linked dimer with apparent subunit molecular weights of 14,000 and 14,500. Binding studies with immobilized botrocetin demonstrated that botrocetin bound to vWF and to a 52/48-kDa region of vWF that contains the GP Ib binding domain, but not to glycocalicin, a proteolytic fragment of GP Ib that contains the vWF binding site. Binding of 125I-labeled vWF to GP Ib-IX complex coated beads and to platelets was strictly botrocetin-dependent with half-maximal binding at a botrocetin concentration of congruent to 0.27 microM. Botrocetin-dependent binding of vWF was specific, saturable, and comparable to that observed with ristocetin. An anti-vWF monoclonal antibody, 3F8, inhibited ristocetin- but not botrocetin-dependent binding of vWF, suggesting the presence of distinct ristocetin and botrocetin modulator sites on vWF. The botrocetin reconstitution assay was at least an order of magnitude more sensitive than the corresponding ristocetin assay for the competitive analysis of functional domains on both vWF and the GP Ib-IX complex and has confirmed the localization of the vWF-binding domain to the 45-kDa N-terminal region of GP Ib.

Amino Acids↗
PubMed1989

Cross-linking of a monomeric 39/34-kDa dispase fragment of von Willebrand factor (Leu-480/Val-481-Gly-718) to the N-terminal region of the alpha-chain of membrane glycoprotein Ib on intact platelets with bis(sulfosuccinimidyl) suberate.

A 39/34-kilodalton (kDa) monomeric dispase fragment of von Willebrand factor (vWF) has been purified by heparin affinity chromatography. Detailed structural analysis of the individual 39- and 34-kDa fragments indicated that they had identical amino acid sequences extending from Leu-480/Val-481 to Gly-718 with an intramolecular disulfide bond between Cys-509 and Cys-695. In addition to the binding site for heparin, the 39/34-kDa fragment also contained binding sites for collagen and for platelet membrane glycoprotein (GP) Ib. Unlike native vWF, the 39/34-kDa fragment bound to GP Ib without the requirement for a modulator but showed increased binding in the presence of botrocetin. The 39/34-kDa vWF fragment was cross-linked to intact human platelets by using the membrane-impermeable, homobifunctional cross-linking reagent bis(sulfosuccinimidyl) suberate. Two distinct cross-linked species of similar molecular weight (220/200 kDa, nonreduced; 190/175 kDa, reduced) were identified by SDS-polyacrylamide gel electrophoresis and autoradiography, consistent with the cross-linking of the 125I-labeled 39/34-kDa vWF fragment to GP Ib. The formation of these cross-linked species was enhanced 1.5-2.5-fold in the presence of the modulator botrocetin. The platelet membrane protein involved in cross-linking was shown unequivocally to be GP Ib since (i) neither cross-linked species was formed with Bernard-Soulier syndrome platelets, which genetically lack the GP Ib-IX complex, (ii) both cross-linked species were specifically immunoprecipitated by anti-GP Ib polyclonal and monoclonal antibodies, and (iii) the formation of the cross-linked species was completely inhibited only by those anti-GP Ib-IX complex monoclonal antibodies that inhibited vWF-GP Ib-IX complex interaction. Proteolysis of cross-linked platelets with endoproteinase Lys-C, which preferentially cleaves off the N-terminal peptide domain on the alpha-chain of GP Ib, indicated that the 39/34-kDa vWF fragment was cross-linked exclusively to this region of the GP Ib-IX complex.

Amino Acid Sequence↗
PubMed1984

Urea and urease.

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Animals↗
PubMedDate not supplied

The use of snake venom toxins as tools to study platelet receptors for collagen and von Willebrand factor.

A large proportion of the biologically active proteins and peptides present within snake venoms interact with components of the haemostatic system to promote or inhibit the normal sequence of events that lead to clot formation. The venom proteins achieve their effects through interaction with various components of the coagulation cascade, endothelial matrix and platelets. Within the latter group, a number of venom proteins target the interaction of platelets with the major adhesive proteins, von Willebrand factor and collagen. The venom proteins bind either the adhesive protein itself or their receptors on the platelet surface, notably GP-Ib-IX-V and GPVI. This review discusses the substantial contribution that venom proteins have made to our understanding of the role of these two adhesive proteins and their receptors (excluding GPIIb-IIIa) in platelet regulation.

Animals↗