Recognition of distinct adhesive sites on fibrinogen by related integrins on platelets and endothelial cells.

Endothelial cells and activated platelets express integrin-type receptors responsible for adhesion to fibrinogen. We have located distinct integrin-directed endothelial cell and platelet attachment sites on immobilized fibrinogen using a combination of synthetic peptides, fibrinogen fragments, and specific anti-peptide monoclonal antibodies. Endothelial cells exclusively recognize an Arg-Gly-Asp-containing site near the C-terminus of the alpha chain (alpha residues 572-574) but fail to recognize the Arg-Gly-Asp sequence in the N-terminal region of the same chain (alpha residues 95-97).

Von Willebrand factor promotes endothelial cell adhesion via an Arg-Gly-Asp-dependent mechanism.

Von Willebrand factor (vWF) is a constitutive and specific component of endothelial cell (EC) matrix. In this paper we show that, in vitro, vWF can induce EC adhesion and promote organization of microfilaments and adhesion plaques. In contrast, human vascular smooth muscle cells and MG63 osteosarcoma cells did not adhere and spread on vWF.

Spontaneous platelet aggregation in type IIB Tampa von Willebrand disease is inhibited by the 52/48-kDa fragment of normal von Willebrand factor, which contains the GPIb binding domain.

The association of Type IIB von Willebrand disease (vWD) with chronic persistent thrombocytopenia and spontaneous platelet aggregation has recently been recognized. It has been shown that IIB von Willebrand factor (vWF) can initiate platelet aggregation by binding to the platelet glycoprotein (GP) lb receptor and inducing exposure of the GpIIb/IIIa fibrinogen receptor. In this study we demonstrate the increased binding of Type IIB Tampa vWF with normal platelets when compared with nonthrombocytopenic Type IIB vWF.

Heparin-associated thrombocytopenia: observations on the mechanism of platelet aggregation.

We investigated the mechanism of heparin-mediated platelet aggregation in 11 patients with heparin-associated thrombocytopenia. Severe thrombocytopenia (16,000 to 66,000 platelets/microliters) developed in each patient during heparin therapy, and platelet aggregation occurred in vitro when heparin was added to mixtures of patient plasma and normal platelet-rich plasma. In 10 patients, heparin-initiated platelet aggregation was inhibited by preincubation of mixtures of normal platelet-rich plasma and heparin-associated thrombocytopenia plasma with monoclonal antiglycoprotein Ib antibodies 6D1 or LJ-Ib1.

Fibrinogen-independent platelet adhesion and thrombus formation on subendothelium mediated by glycoprotein IIb-IIIa complex at high shear rate.

Platelet adhesion and thrombus formation on subendothelium, studied at a shear rate of 2,600 s-1, were inhibited by two synthetic peptides known to interact with GPIIb-IIIa. One peptide (HHLGGAKQAGDV) corresponds to the carboxyl terminal segment of the fibrinogen gamma-chain (gamma 400-411) and the other (RGDS) contains the amino acid sequence Arg-Gly-Asp (RGD) common to fibronectin, von Willebrand factor, vitronectin and the alpha-chain of fibrinogen. Neither platelet adhesion nor thrombus formation were decreased in a patient with severe congenital fibrinogen deficiency and this was equally true when his blood was further depleted of the small amounts of fibrinogen present utilizing an anti-fibrinogen antibody.

Isolation and functional characterization of the von Willebrand factor-binding domain located between residues His1-Arg293 of the alpha-chain of glycoprotein Ib.

In the present report we describe the isolation of a functional domain of platelet membrane glycoprotein (GP) Ib which retains von Willebrand factor (vWF)-binding activity. Glycocalicin, a proteolytic fragment of the alpha-chain of GP Ib generated by an endogenous calcium-activated protease, was submitted to digestion with trypsin. The two resulting fragments, one of 45 kDa extending between residues His1 and Arg293 and representing the amino terminus of the alpha-chain, the other of 84 kDa corresponding to the previously described macroglycopeptide, were purified to homogeneity.

Structure of the von Willebrand factor domain interacting with glycoprotein Ib.

von Willebrand factor is a multifunctional adhesive protein of plasma, platelets, and endothelial cells that mediates a crucial interaction for normal hemostasis and thrombus formation by binding to platelet membrane glycoprotein Ib. We provide here evidence that this function involves two limited noncontiguous regions of the molecule, each contained within 15 amino acid residues, separated in the linear sequence by 205 residues, and maintained in close spatial proximity in the folded molecule by disulfide bonding. Definition of this chemical structure clarifies a fundamental mechanism of platelet adhesion to thrombogenic surfaces and sets the bases for obtaining synthetic replicas that may be used to modulate platelet function.

Generation and characterization of specific antibodies directed to the binding domain of adhesive molecules.

Antibodies were selected in rabbits using as immunogens five synthetic peptides representing the putative cell-binding domains of von Willebrand factor (vWF), fibrinogen (Fn), fibronectin (Fn) and vitronectin. All peptide immunogens contained the sequence Arg-Gly-Asp, thought to be involved in ligand binding to platelet glycoprotein (GP) IIb-IIIa; two of them corresponded to two distinct regions in the alpha-chain of fibrinogen. All anti-peptide antibodies recognized the corresponding immunogen, and four of them, i.

Generation and characterization of peptide-specific antibodies that inhibit von Willebrand factor binding to glycoprotein IIb-IIIa without interacting with other adhesive molecules. Selectivity is conferred by Pro1743 and other amino acid residues adjacent to the sequence Arg1744-Gly1745-Asp1746.

We have generated antibodies against a synthetic peptide corresponding to the sequence of human von Willebrand factor (vWF) between residues Glu1737-Ser1750 which includes the Arg-Gly-Asp sequence common to several adhesive molecules. Two anti-peptide antibodies, one polyclonal, and one monoclonal reacted with native vWF and inhibited its binding to platelet glycoprotein (GP) IIb-IIIa, but showed negligible cross-reactivity with fibrinogen, fibronectin, and vitronectin, three other molecules that contain the sequence Arg-Gly-Asp and bind to platelets. The structural bases for the specificity of the two antibodies were evaluated by testing the ability of peptides homologous to the parent sequence, but with single amino acid substitutions, to neutralize the binding of the two antibodies to vWF.

Homozygous and heterozygous deletions of the von Willebrand factor gene in patients and carriers of severe von Willebrand disease.

Severe von Willebrand disease is characterized by undetectable or trace quantities of von Willebrand factor in plasma and tissue stores. We have studied the genomic DNA of 10 affected individuals from six families with this disorder using probes from the 5' and 3' ends of the vWF cDNA and with a probe extending from the 5' end into the central region. Southern blots of restriction endonuclease digests and gene dosage analysis measurements carried out with quantitative slot blots of undigested genomic DNA separated these patients into three groups.

Effects of monoclonal antibodies against the platelet glycoprotein IIb/IIIa complex on thrombosis and hemostasis in the baboon.

To assess the hemostatic consequences and antithrombotic effectiveness of blocking the platelet glycoprotein (GP) IIb/IIIa receptor for fibrinogen and other adhesive glycoproteins in vivo, well characterized murine monoclonal antibodies against the platelet GP IIb/IIIa complex, AP-2 and LJ-CP8, were infused intravenously into baboons. Four animals each received doses of 0.2, 0.

Unique interactions of asialo von Willebrand factor with platelets in platelet-type von Willebrand disease.

The present studies demonstrate that platelets from patients with platelet-type von Willebrand disease show specific and saturable binding of asialo von Willebrand factor (AS-vWF) under conditions where such binding is not observed with normal platelets. Although specific binding of 125I-AS-vWF to formalin-fixed normal platelets could not be demonstrated, specific binding to fixed patient platelets was seen with an apparent Kd of 1.3 micrograms/mL and specific maximally bound ligand of 0.

Isolation and characterization of two domains of human von Willebrand factor that interact with fibrillar collagen types I and III.

We have identified four discrete proteolytic fragments of von Willebrand factor (vWF) that define two collagen-binding domains. Two of the fragments tested, T 96 kDa and T 55 kDa, were generated by digestion with trypsin, and two, Fragments I and III, with Staphylococcus aureus V8 protease. The larger Fragment III, a disulfide-linked homodimer, extends between residues 1 and 1365 of the 2050-residue vWF subunit and comprises the sequence of all the others.

The von willebrand factor domain-mediating botrocetin-induced binding to glycoprotein IB lies between Val449 and Lys728.

Botrocetin, a component of Bothrops jararaca venom, induces von Willebrand factor (vWF)-dependent platelet agglutination and has been proposed as an alternative agent to ristocetin for evaluating vWF function. However, important differences between the vWF-platelet interactions induced by these two agents have suggested that different regions of vWF and the platelet may be involved in the interactions induced by the two agonists. We have recently demonstrated that binding of vWF to the platelet glycoprotein (GP) Ib receptor, either induced by ristocetin or as occurs spontaneously with asialo-vWF or vWF from IIb von Willebrand disease, is mediated by a domain residing on a 52/48-kilodalton (kD) tryptic fragment of vWF.

von Willebrand factor and von Willebrand disease.

Progress has occurred in the past several years in the understanding of the structure and function of von Willebrand factor (vWF). This multimeric glycoprotein exhibits a dual role, that of mediating platelet adhesion and aggregation onto thrombogenic surfaces, and that of functioning as carrier in plasma for the factor VIII procoagulant protein. New insights into the nature of the several functional domains of vWF have led to the identification of the regions of the molecule that interact with factor VIII, heparin, the glycoprotein lb of platelets, and collagen.

Increased surface expression of the membrane glycoprotein IIb/IIIa complex induced by platelet activation. Relationship to the binding of fibrinogen and platelet aggregation.

Platelet activation altered the binding of three monoclonal antibodies (monovalent Fab' fragment) directed against the glycoprotein (GP) IIb/IIIa complex. An increased binding of two- to threefold occurred after stimulation with thrombin or phorbol myristate acetate (PMA), with slight but significant increase in the dissociation constants (Kd) of two antibodies (LJ-CP8 and LJ-P9). In contrast, no statistically significant changes were observed with ADP-stimulated platelets.

Amino acid sequence of the von Willebrand factor-binding domain of platelet membrane glycoprotein Ib.

We report the amino acid sequence of a 299-residue segment from the alpha chain of the human platelet membrane glycoprotein Ib. This includes the complete sequence of the amino-terminal tryptic fragment of 290 residues comprising the von Willebrand factor-binding domain. Two primary sets of overlapping fragments were obtained by cleavage of the S-carboxymethylated protein at methionyl and lysyl bonds following treatment with cyanogen bromide and Achromobacter protease I, respectively.

Type IIB von Willebrand factor with normal sialic acid content induces platelet aggregation in the absence of ristocetin. Role of platelet activation, fibrinogen, and two distinct membrane receptors.

Three preparations of purified von Willebrand factor (vWF), obtained from unrelated patients affected by type IIB von Willebrand disease, were found to have normal sialic acid content (between 129 and 170 nmol/mg of vWF, as compared with 158 +/- 17 nmol/mg in four normal preparations) and to induce platelet aggregation in the presence of physiologic levels of divalent cations and without addition of ristocetin. A monoclonal antibody that blocks the vWF binding domain of the platelet glycoprotein (GP)Ib caused complete inhibition of IIB vWF-induced aggregation. In contrast, a monoclonal antibody that blocks the receptor for adhesive proteins on the platelet GPIIb/IIIa complex failed to inhibit the initial response of platelets to high concentrations of IIB vWF.

Proteolytic degradation of von Willebrand factor after DDAVP administration in normal individuals.

The infusion of 1-deamino-8-D-arginine vasopressin (DDAVP) in normal individuals is followed by an increase in factor VIII/von Willebrand factor (vWF) in plasma, by an increase in intensity of all sizes of multimers, and by the appearance of larger multimers of vWF than those seen in the resting state. Since the larger multimers are rapidly cleared and proteolysis is known to cause disaggregation of large multimers, we evaluated the degree of vWF proteolysis after DDAVP administration. DDAVP was infused into eight normal adult volunteers, and the relative proportions of the intact 225 kilodalton (kDa) subunit and the 189, 176, and 140 kDa vWF fragments were compared before and at different times after DDAVP infusion.

A heparin-binding domain of human von Willebrand factor. Characterization and localization to a tryptic fragment extending from amino acid residue Val-449 to Lys-728.

We have recently shown that the domain of von Willebrand factor (vWF) which interacts with the platelet glycoprotein Ib (GPIb) is located in a 52/48-kDa tryptic fragment of the molecule which begins with amino acid residue Val-449. We have now established that the fragment extends to residue Lys-728 and demonstrate here that a high affinity heparin-binding domain of vWF also lies within this region and in close proximity to that for GPIb. We have used an assay employing heparin coupled to Sepharose CL-6B to show that 125I-vWF binds to heparin in a time-dependent, saturable, and reversible manner.

von Willebrand disease.

von Willebrand disease (vWD) is a bleeding disorder characterized by a complex hemostatic defect. Abnormal platelet function, usually reflected by a prolonged bleeding time, is the result of a quantitative or qualitative defect of von Willebrand factor (vWF). A secondary deficiency of factor VIII procoagulant protein (factor VIII) may occur leading to a coagulation defect as well.

Epitope mapping of the von Willebrand factor subunit distinguishes fragments present in normal and type IIA von Willebrand disease from those generated by plasmin.

A small but consistent proportion of the von Willebrand factor (vWF) in normal plasma is composed of 189, 176, and 140 kD fragments cleaved from the 225 kD subunit. A monoclonal antibody map of vWF, based on the reactivity of individual antibodies with cyanogen bromide and tryptic fragments of known carboxy and/or amino termini, showed that in normal and IIA von Willebrand disease (vWD) plasmas the 140 kD fragment was derived from the amino-terminal region, whereas the 176 kD fragment was derived from the carboxy-terminal region of the subunit. In type IIA vWD, however, the fragments comprised a greater proportion of circulating vWF.

Subunit composition of plasma von Willebrand factor in patients with the myeloproliferative syndrome.

In order to evaluate the role of proteolysis in acquired von Willebrand's disease (vWD) associated with the myeloproliferative syndrome, we have determined the relative quantity of von Willebrand factor (vWF) fragments as compared with the intact 225 kDa subunit in four patients. The plasma vWF of each individual lacked large multimers; each had a prolonged bleeding time; and both platelet and leukocyte counts were elevated. Plasma was obtained from blood drawn into 1 mmol/L leupeptin, 6 mmol/L N-ethylmaleimide, and 5 mmol/L EDTA to prevent in vitro proteolysis.