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Z M Ruggeri

Publications and source records attributed to Z M Ruggeri.

At least 73 records · Page 4Linked to original sources
PubMed1995

Identification of three tyrosine residues of glycoprotein Ib alpha with distinct roles in von Willebrand factor and alpha-thrombin binding.

The interaction between von Willebrand factor (vWF) and the platelet membrane glycoprotein (GP) Ib-IX-V complex is essential for platelet adhesion at sites of vascular injury under high shear stress flow conditions. Moreover, GP Ib-IX-V may contribute to the mechanisms of platelet activation through its high affinity binding of alpha-thrombin. There are two distinct but partially overlapping regions of GP Ib alpha thought to be involved in interacting with vWF (residues 251-279) and alpha-thrombin (residues 271-284); they share three tyrosine residues (positions 276, 278, and 279) that have recently been shown to be sulfated (Dong, J., Li, C. Q., and Lopez, J.A. (1994) Biochemistry 33, 13946-13953). To define the functional role of these three residues, we have introduced selected mutations in a soluble recombinant GP Ib alpha fragment (corresponding to the sequence 1-302 of the mature protein) that binds vWF and alpha-thrombin with the same attributes as intact GP Ib-IX-V complex. Fragments containing a single Tyr-->Phe substitution either at position 276 or 278 or 279 exhibited normal interaction with vWF but markedly reduced or absent binding of alpha-thrombin. GP Ib alpha fragment with normal sequence but synthesized under sulfate-free conditions also failed to bind alpha-thrombin and, in addition, had markedly reduced interaction with vWF. The simultaneous substitution of three neighboring Asp residues with Asn at positions 272, 274, and 277, a multiple mutation that may impair Tyr sulfation, also resulted in loss of binding of both ligands. These results define distinct structural features of GP Ib alpha selectively involved in supporting the interaction with vWF or alpha-thrombin.

Animals↗
PubMed1995

Von Willebrand's disease and the mechanisms of platelet function.

Von Willebrand's disease, the most common congenital bleeding disorder in humans, is the consequence of quantitative and/or qualitative defects of von Willebrand factor, a protein necessary for platelet adhesion and thrombus formation at sites of vascular injury. The definition of the molecular basis of von Willebrand's disease has helped clarify the structure of von Willebrand factor as well as its essential role in platelet function, particularly under haemodynamic conditions of high shear stress. Platelets respond rapidly to alterations of endothelial cells by attaching firmly to the site of lesion, where exposure of subendothelial components may have occurred. The first layer of platelets is in contact with the thrombogenic surface (adhesion), whereas subsequent growth of the haemostatic plug depends on platelet-platelet interactions (aggregation). Both aspects of platelet function are influenced by von Willebrand factor binding to specific platelet membrane receptors as well as subendothelial structures, such as collagen.

Blood Platelets↗
PubMed1995

The role of von Willebrand factor and fibrinogen in the initiation of platelet adhesion to thrombogenic surfaces.

Platelets respond rapidly to alterations of endothelial cells by attaching firmly to the site of lesion, where exposure of subendothelial components may have occurred. The first layer of platelets is in contact with the thrombogenic surface (adhesion), whereas subsequent growth of the hemostatic plug depends on platelet-to-platelet interactions (aggregation). Both aspects of platelet function are influenced by von Willebrand factor and fibrinogen interaction with specific platelet membrane receptors. Multiple domains of von Willebrand factor and fibrinogen are involved in securing initiation and growth of platelet thrombi.

Endothelium, Vascular↗
PubMed1995

Two antipeptide monoclonal antibodies that recognize adhesive sequences in fibrinogen: identification of antigenic determinants and unrelated sequences using synthetic combinatorial libraries.

The fine specificity of two different monoclonal antibodies raised against synthetic peptides, each representing one of the two Arg-Gly-Asp (RGD) sequences in fibrinogen, was examined using synthetic combinatorial libraries (SCLs). The monoclonal antibodies (mAb), mAb LJ-134B/29 and mAb LJ-155B/16, recognize both the immunogenic peptide and native fibrinogen. The specificity of mAb LJ-134B29 was mapped using hexa- and decapeptide positional scanning SCLs (PS-SCLs) and competitive ELISA. The most active amino acids at each position of the two libraries were identified from a single screening. Individual hexa- and decapeptides were synthesized and assayed to determine their binding affinities. The 16 individual hexapeptides represented single and multiple substitutions of the antigenic determinant sequence, -GDSTFE-, eight of which had affinities less than 10nM. Four of the twelve individual decapeptides were found to have binding affinities of approximately 300nM, or nearly three-fold less than the peptide immunogen. A dual-defined hexapeptide library was screened against mAb LJ-155B/16, and individual peptides were obtained through an iterative selection and synthesis process. Surprisingly, one of the most active sequences was Ac-WWYESW-NH2 (IC50 = 40nM), which showed no similarity to the sequence of the immunizing peptide. Further mapping of the specificity of this antibody revealed that the antigenic determinant within the peptide immunogen was not completely linear. Recognition of this unrelated sequence by mAb LJ-155B/16 was confirmed in a direct binding assay using biotinylated peptide. The use of SCLs for the elucidation of high affinity peptides recognized by these two antibodies may provide additional information on the molecular mechanisms of fibrinogen binding to different integrin receptors.

Amino Acid Sequence↗
PubMed1994

Proteolysis of von Willebrand factor in therapeutic plasma concentrates.

Therapeutic plasma concentrates containing vo Willebrand factor (vWF) lack the largest, most hemostatically active multimers. To evaluate whether this abnormality results from proteolysis during manufacturing, we have analyzed the subunit structure of vWF in several commercial products and found a marked reduction in the relative content of intact 225-kD subunit, paralleled by an increase in the proteolytic fragments normally present in plasma, particularly that of 176 kD. There was no heightened vWF fragmentation in blood-bank cryoprecipitate prepared from platelet-poor, single-donor plasma; in contrast, there was a marked degree of fragmentation in cryoprecipitate prepared from pooled plasmapheresis plasma representing the starting fraction for the production of commercial concentrates. In cryoprecipitate prepared experimentally from plasma containing varying numbers of platelets, the degradation of vWF was proportional to the platelet count, but was greatly diminished by adding protease inhibitors to the plasma. On the basis of these findings, we postulate that the loss of the largest vWF multimers in commercial products results from the use of poorly centrifuged plasmapheresis plasma containing an excessive number of residual platelets and leukocytes. These cells, lysing when plasma is frozen and thawed for the preparation of cryoprecipitate, may liberate proteolytic enzymes that cleave the vWF subunit and contribute to the degradation of the largest multimers. Our results should help devise new approaches for the preparation of more effective concentrates for the treatment of von Willebrand disease.

Blood Platelets↗
PubMed1994

Localization and characterization of an alpha-thrombin-binding site on platelet glycoprotein Ib alpha.

Glycoprotein (GP) Ib alpha is required for expression of the highest affinity alpha-thrombin-binding site on platelets, possibly contributing to platelet activation through a pathway involving cleavage of a specific receptor. This function may be important for the initiation of hemostasis and may also play a role in the development of pathological vascular occlusion. We have now identified a discrete sequence in the extracytoplasmic domain of GP Ib alpha, including residues 271-284 of the mature protein, which appears to be part of the high affinity alpha-thrombin-binding site. Synthetic peptidyl mimetics of this sequence inhibit alpha-thrombin binding to GP Ib as well as platelet activation and aggregation induced by subnanomolar concentrations of the agonist; they also inhibit alpha-thrombin binding to purified glycocalicin, the isolated extracytoplasmic portion of GP Ib alpha. The inhibitory peptides interfere with the clotting of fibrinogen by alpha-thrombin but not with the amidolytic activity of the enzyme on a small synthetic substrate, a finding compatible with the concept that the identified GP Ib alpha sequence interacts with the anion-binding exosite of alpha-thrombin but not with its active proteolytic site. The crucial structural elements of this sequence necessary for thrombin binding appear to be a cluster of negatively charged residues as well as three tyrosine residues that, in the native protein, may be sulfated. GP Ib alpha has no significant overall sequence homology with the thrombin inhibitor, hirudin, nor with the specific thrombin receptor on platelets; all three molecules, however, possess a distinct region rich in negatively charged residues that appear to be involved in thrombin binding. This may represent a case of convergent evolution of unrelated proteins for high affinity interaction with the same ligand.

Adenosine Triphosphate↗
PubMed1994

Bernard-Soulier syndrome: quantitative characterization of megakaryocytes and platelets by flow cytometric and platelet kinetic measurements.

Platelets and megakaryocytes have been characterized in a Bernard-Soulier syndrome (BSS) kindred with respect to glycoprotein (GP) membrane receptors and measurements of thrombocytopoiesis. The index patient exhibited lifelong bleeding tendency, moderate thrombocytopenia (35 x 10(9)/l), giant platelets (mean platelet volume 12.5 microns 3 compared to 7.5 +/- 1.5 microns 3 in normals), absent ristocetin-induced platelet agglutination and absent binding of von Willebrand factor (vWF). Flow-cytometric analysis revealed absent platelet binding (0-2%) of monoclonal antibodies (mAb, LJ-P3, LJ-Ib1 and LJ-Ib10) directed against distinct epitopes on membrane GPIb alpha of the GPIb-IX complex, and normal binding of LJ-P4 mAb directed against GPIIb/IIIa complex (relative to increased platelet surface area). Marrow megakaryocytes also failed to express GPIb-IX complex, but demonstrated normal expression of GPIIb/IIIa. Among 6 asymptomatic family members, the patient's mother and 2 of his 4 children exhibited approximately 50% binding of anti-GPIb alpha mAb to their platelets by both flow cytometry and direct binding studies using 125I-vWF, 125I-LJ-Ib1 and 125I-LJ-Ib10 mAb. Marrow megakaryocytes were increased in the average cell volume and cytoplasmic granularity with a corresponding increase in ploidy (46% > 16N compared to 22 +/- 5% in normal individuals), a pattern typical of megakaryocytes stimulated by thrombocytopenia. Autologous 111In-platelet life span was shortened to 4.1 days (compared with 9.5 +/- 0.5 days in normal subjects), and the turnover of platelet mass in the circulation was near normal. The data directly demonstrate that the platelet membrane GPIb-IX defect in BSS originates in megakaryocytes at all levels of cell maturation, and exclude the possibility that the receptor abnormality is acquired during cell maturation or after platelets are released into the circulation. Since marrow megakaryocytes exhibited cellular changes consistent with stimulated megakaryocytopoiesis, these results also suggest that thrombocytopenia in this kindred of BSS is a consequence of both decreased platelet survival and ineffective platelet production.

Adult↗
PubMed1993

Conformational changes in fibrinogen elicited by its interaction with platelet membrane glycoprotein GPIIb-IIIa.

The binding of fibrinogen to membrane glycoprotein GPIIb-IIIa on activated platelets leads to platelet aggregation. This interaction results in conformational changes in fibrinogen as evidenced by the expression of receptor-induced binding sites, RIBS, epitopes which are expressed by the bound but not the free ligand. In the present study, two RIBS epitopes have been localized. One sequence resides at gamma 112-119 and is recognized by mAb 9F9; the second is the RGDF sequence at A alpha 95-98 and is recognized by mAb 155B16. These epitopes are also exposed by adsorption of fibrinogen onto a plastic surface and digestion of the molecule by plasmin. Proteolytic exposure of the epitopes coincides with cleavage of the carboxyl-terminal aspects of the A alpha-chains to form fragment X2. The inaccessibility of the RGDF sequence at A alpha 95-98 in fibrinogen suggests that this sequence does not participate in the initial binding of the molecule to GPIIb-IIIa. The location of these RIBS epitopes suggests a model in which binding of fibrinogen to its receptor alters the conformation of the carboxyl-terminal aspects of the A alpha-chains, exposing the sequences which reside in the coiled-coil connector segments between the D and E domains of the molecule. These sequences may then serve as epitopes and may mediate unique functions of the receptor-bound molecule.

Amino Acid Sequence↗
PubMed1993

Elevated platelet count as a cause of abnormal von Willebrand factor multimer distribution in plasma.

Twelve of 19 patients with myeloproliferative disorders showed a decrease of absence of the largest multimers of plasma von Willebrand factor (vWF) that correlated with elevated platelet counts but not with leukocyte counts. This suggested that platelets, rather than leukocytes, may be associated with the pathogenesis of the acquired vWF abnormality seen in these patients. To examine the hypothesis further, we studied 12 patients with reactive thrombocytosis after splenectomy. Increased platelet count (> 5 x 10(11)/L) after splenectomy was associated with vWF abnormalities indistinguishable from those detected in patients with myeloproliferative disorders. Accordingly, there was an inverse correlation between proportion of large vWF multimers and platelet, but not leukocyte, number: normalization of the platelet count was accompanied by restoration of a normal vWF multimeric pattern. These findings suggest that an increase in the number of platelets circulating in blood may favor the adsorption of larger vWF multimers onto the platelet membrane, resulting in their removal from the circulation and subsequent degradation.

Adult↗
PubMed1993

Platelet aggregation on extracellular matrix: effect of a recombinant GPIb-binding fragment of von Willebrand factor.

Platelets in whole blood incubated on extracellular matrix (ECM) produced by bovine corneal endothelial cells under oscillatory flow conditions demonstrate extensive aggregate formation. Since both platelet-subendothelium and platelet-platelet interactions are mediated by von Willebrand factor (vWF), we used this system to examine the effect of a recombinant GPIb-binding fragment of vWF (designated RG12986), comprising residues 445-733 of the native vWF subunit, on platelet reactivity with ECM. The seven cysteines present in the RG12986 fragment were reduced and alkylated in order to achieve a monomeric conformation. The recombinant vWF fragment binds to unstimulated platelets in the absence of exogenous modulators. When added to platelet-rich plasma, it inhibits ristocetin-induced platelet agglutination. Binding of 51Cr-labeled platelets in reconstituted whole blood to ECM was inhibited by RG12986 in a dose dependent and saturable manner, with IC50 of 4 microM and maximal inhibition (about 70%) at 6 microM. Scanning electron microscope (SEM) analysis showed that addition of RG12986 to whole blood significantly inhibited platelet aggregation on ECM. The extent of inhibition observed with RG12986 at a final concentration of 4 microM was similar to that obtained with the cell adhesion peptide RGDS at the concentration of 0.1 mM. The ability of the RG12986 fragment to inhibit platelet aggregation on ECM is in agreement with the concept that blockade of vWF-GPIb interaction may inhibit further events leading to activation of the glycoprotein IIb/IIIa (GPIIb/IIIa) complex and subsequent thrombus formation.

Amino Acid Sequence↗
PubMed1993

Analysis of structure-function relationships in the platelet membrane glycoprotein Ib-binding domain of von Willebrand's factor by expression of deletion mutants.

We have used a series of Escherichia coli-expressed deletion mutants of the glycoprotein (GP) Ib-binding domain of von Willebrand factor (vWF) to study the structural basis of its function. In addition to the prototypic molecule (rvWF441-733), we constructed 11 mutants; seven had deletions of sequence on the amino- and/or carboxyl-terminal side of the Cys509-Cys695 intrachain disulfide loop, and four had limited deletions inside the loop. Other cysteine residues in addition to 509 and 695, when present in the corresponding native sequence, were mutated to glycine; all molecules were purified in the oxidized as well as reduced and alkylated state. The smallest species retaining the ability to interact with GP Ib in the absence of modulators was the oxidized rvWF508-696; the latter, as well as rvWF441-696, became inactive after reduction and alkylation. In contrast, all the other fragments with deletions outside of the loop, but extending at least to residue 700, showed better binding to platelets after reduction and alkylation than when the Cys509-Cys695 disulfide bond was oxidized. Any limited deletion of sequence inside the loop caused complete loss of GP Ib-binding function both in the absence or in the presence of botrocetin, and this persisted even after reduction and alkylation. In contrast, all mutants with intact sequence between residues 509 and 695 bound to GP Ib in the presence of botrocetin, regardless of whether the 2 cysteine residues were oxidized or reduced and alkylated. Ristocetin, unlike botrocetin, appeared to have no effect in modulating the binding of any of the expressed fragments to platelets. Our findings suggest that the GP Ib-binding domain of vWF contains multiple interaction sites, but integrity of the sequence 509-695 is important for function.

Amino Acid Sequence↗
PubMed1993

Expression of human platelet glycoprotein Ib alpha in transgenic mice.

Platelets are cytoplasmic fragments of megakaryocytes and, therefore, their membrane proteins cannot be manipulated by expression methods in culture. To overcome this limitation, we have expressed human glycoprotein (GP) Ib alpha in transgenic mouse megakaryocytes and found that it was present on the surface of platelets associated with the mouse GP Ib beta subunit. This finding demonstrates that assembly of the heterooligomeric GP Ib complex occurs through mechanisms conserved across species. In contrast, the receptor function of GP Ib exhibited restricted species specificity, since only the chimeric complex containing human GP Ib alpha bound human von Willebrand factor and supported platelet aggregation mediated by ristocetin. These studies demonstrate the transgenic engineering of a platelet adhesion receptor under control of the human GP Ib alpha promoter and illustrate a new approach to manipulate platelet receptors and study structure-function relationships in hemostasis and thrombosis.

Animals↗
PubMed1993

Real-time analysis of shear-dependent thrombus formation and its blockade by inhibitors of von Willebrand factor binding to platelets.

Two likely mechanisms for the initiation of arterial platelet thrombus formation under conditions of elevated fluid shear stresses are: (1) excessive adhesion and aggregation of platelets from rapidly flowing blood onto the exposed sub-endothelium of injured, atherosclerotic arteries; or (2) direct, fluid shear stress-induced aggregation of platelets in constricted arteries with intact endothelial cells. Mechanism (1) was simulated using a parallel plate flow chamber, fibrillar collagen type I-coated slides, and mepacrine-labeled (fluorescent) platelets in whole blood anticoagulated with citrate, hirudin, unfractionated porcine heparin, or low molecular weight heparin flowing for 1 to 2 minutes at wall shear rates of 100 to 3,000 seconds-1 (4 to 120 dynes/cm2). The precise sequence of interactions among von Willebrand factor (vWF), glycoprotein (GP)Ib, and GPIIb-IIIa during platelet adhesion and subsequent aggregation were resolved by direct real-time observation using a computerized epifluorescence video microscopy system. Adhesion at high shear rates was the result of the adsorption of large vWF multimers onto collagen and the binding of platelet GPIb to the insolubilized vWF. Aggregation occurred subsequently and required the binding of ligands, including vWF via its RGD binding domain, to GPIIb-IIIa. Mechanism (2) was modeled by producing shear stresses of 90 to 180 dynes/cm2 in a rotational cone and plate viscometer, which aggregates platelets from platelet-rich-plasma (PRP) anti-coagulated with citrate, hirudin, or either type of heparin in reactions that require large vWF multimers, Ca2+, adenosine diphosphate, and both GPIb and GPIIb-IIIa. Both vWF-mediated shear-aggregation in PRP and platelet-collagen adhesion in flowing whole blood (anticoagulated with citrate and hirudin) are inhibited by two potentially useful anti-arterial thrombotic agents: polymeric aurin tricarboxylic acid (ATA; 28.5 to 114 micrograms/mL), which binds to vWF and inhibits its attachment of GPIb, and a recombinant vWF fragment (rvWF445-733; 30 to 200 micrograms/mL) that binds to platelet GPIb (in the absence of any modulator) and blocks attachment of vWF multimers. Unfractionated heparin, but not low molecular weight heparin, apparently binds to rvWF445-733 and counteracts the inhibitory effects of the vWF fragment in vitro on shear-aggregation and platelet-collagen adhesion.

Antibodies, Monoclonal↗
PubMed1993

Disulfide bond requirements for assembly of the platelet glycoprotein Ib-binding domain of von Willebrand factor.

von Willebrand factor (vWF) is a multimeric glycoprotein of plasma and the subendothelial matrix that interacts with specific platelet receptors to establish platelet adhesion at a site of vascular injury. The vWF domain containing the platelet receptor glycoprotein Ib-binding site can be expressed in heterologous cells as a recombinant homodimeric fragment that mediates platelet-platelet interaction, analogous to multimeric vWF. The recombinant domain, r116, contains 7 Cys residues within its 290-residue monomeric subunit paired in an unidentified intra- and intermolecular disulfide bond arrangement. In this report we define the disulfide bond-dependent framework of r116 that provides the domain with its essential structural features that support dimer formation and the generation of a disulfide bond-dependent epitope. The results demonstrate that a triplet of Cys residues at positions 459, 462, and 464 are essential for efficient dimer formation. An intramolecular Cys509/Cys695 disulfide loop is required for generating a functional dimeric molecule, and monomeric molecules containing a Cys509/Cys695 intramolecular disulfide bond are unable to support ristocetin-mediated platelet aggregation. The disulfide arrangement in r116 is similar, if not identical, to the proposed arrangement within the corresponding region of plasma vWF, and these studies document the inherent Cys-dependent maturation of an isolated vWF domain.

Disulfides↗
PubMed1993

von Willebrand factor.

von Willebrand factor is a multimeric glycoprotein essential for the normal arrest of bleeding after tissue injury (hemostasis). The molecule is present in blood, both in plasma and inside platelets, as well as in endothelial cells and the subendothelial matrix of the vessel wall. Through multiple functional domains, von Willebrand factor mediates the attachment of platelets to exposed tissues, where discontinuity of the vascular endothelium occurs, and the subsequent platelet aggregation leading to the formation of platelet thrombi. The crucial role of von Willebrand factor in platelet function is particularly apparent when hemodynamic conditions create blood flow with high shear stress, as in capillaries under physiologic conditions or in stenosed and partially occluded arteries in disease states. The involvement of von Willebrand factor in the processes that lead to acute thrombosis has attracted considerable interest on the molecular and functional biology of the protein. Indeed, understanding the mechanisms and structural bases for von Willebrand factor function may result in new and effective approaches to anti-thrombotic intervention.

Amino Acid Sequence↗