Mapping the glycoprotein Ib-binding site in the von willebrand factor A1 domain.

The von Willebrand factor (vWF) mediates platelet adhesion to exposed subendothelium at sites of vascular injury. It does this by forming a bridge between subendothelial collagen and the platelet glycoprotein Ib-IX-V complex (GPIb). The GPIb-binding site within vWF has been localized to the vWF-A1 domain. Based on the crystal structure of the vWF-A1 domain (Emsley, J., Cruz, M., Handin, R., and Liddington, R. (1998) J. Biol. Chem. 273, 10396-10401), we introduced point mutations into 16 candidate residues that might form all or part of the GPIb interaction site. We also introduced two mutations previously reported to impair vWF function yielding a total of 18 mutations. The recombinant vWF-A1 mutant proteins were then expressed in Escherichia coli, and the activity of the purified proteins was assessed by their ability to support flow-dependent platelet adhesion and their ability to inhibit ristocetin-induced platelet agglutination. Six mutations located on the front and upper anterior face of the folded vWF-A1 domain, R524S, G561S, H563T, T594S/E596A, Q604R, and S607R, showed reduced activity in all the assays, and we suggest that these residues form part of the GPIb interaction site. One mutation, G561S, with impaired activity occurs in the naturally occurring variant form of von Willebrand's disease-type 2M underscoring the physiologic relevance of the mutations described here.

Expression and activation of protein kinase C isoforms in a human megakaryocytic cell line.

Megakaryocytes undergo a unique differentiation program, becoming polyploid through repeated cycles of DNA synthesis without concomitant cell division. We have shown previously that phorbol 12-myristate 13-acetate (PMA) induces the Dami human megakaryocytic cell line to become polyploid and to express platelet-specific proteins, including von Willebrand factor (vWF) and glycoprotein Ib (GpIb). Phorbol esters are thought to regulate gene expression principally through the activation of protein kinase C (PKC), a family of structurally related kinases with potentially unique activation requirements and substrate specificities. A survey of PKC isoforms in Dami cells revealed that, by both Western and Northern analyses, PKC isoforms alpha, beta, delta, epsilon, eta, theta, and zeta were reproducibly detected. PKC-gamma was not detected. In order to define the role of individual PKC isoforms in megakaryocytic maturation, PMA and 2-deoxyphorbol 13-phenylacetate 20-acetate (dPPA), a putative selective activator of the PKC-beta 1 isotype, were compared for their effects on Dami cell maturation. Treatment with either dPPA or PMA caused Dami cells to cease proliferating, to become polyploid, and to express vWF. We also examined dPPA and PMA for their ability to activate and to downregulate expression of different PKC isoforms. Fifteen-minute treatment with PMA resulted in the translocation of PKC isoforms alpha, epsilon, and theta from the cytosolic to the membrane fraction; twenty-four hour treatment resulted in the downregulation of these isoforms. In contrast, dPPA was found to be a potent activator of PKC-epsilon alone and exhibited weaker effects on alpha and theta. These data suggest that PKC isoforms beta, delta, eta, and zeta, which appear not to be activated by either phorbol ester, are unlikely to be primarily involved in megakaryocytic maturation in response to these agents. The isoforms that are translocated by both phorbol esters-PKC isoforms alpha and theta, and particularly epsilon-are more likely to transduce the signals that stimulate Dami cell differentiation.

Platelet adhesion to multimeric and dimeric von Willebrand factor and to collagen type III preincubated with von Willebrand factor.

As part of a systematic study of platelet interaction with adhesive proteins under flow conditions, we studied platelet adhesion to multimeric and dimeric von Willebrand factor (vWF) coated to glass. vWF-dependent adhesion to collagen type III was studied for comparison. Adhesion to glass-coated vWF and vWF-mediated adhesion to collagen type III were in many respects similar. Both showed no decrease at increasing shear rates and a decline to 50% of maximum with a low-molecular-weight multimeric fraction. Adhesion to glass-coated vWF was partially inhibited by heparin and completely inhibited by prostaglandin I(2) and anti-glycoprotein (GP) Ib and anti-GPIIb-IIIa antibodies. vWF-dependent adhesion to collagen was not inhibited by heparin, was partially inhibited by anti-GPIIb-IIIa, and was completely inhibited by prostaglandin I(2) and anti-GPIb. Recombinant dimeric vWF was made by deletion of the propeptide and expression in Chinese hamster ovary cells. Adhesion was 50% of that with plasma vWF, and larger concentrations of dimeric vWF were required. Adhesion to dimeric vWF was optimal at 1500 s(-1), with a gradual decrease at higher shear rates. We conclude that adhesion to collagen type III is strongly but not completely determined by the adhesive properties of vWF.

Interaction of the von Willebrand factor (vWF) with collagen. Localization of the primary collagen-binding site by analysis of recombinant vWF a domain polypeptides.

The von Willebrand factor (vWF) mediates platelet adhesion to the vascular subendothelium by binding to collagen, other matrix constituents, and the platelet receptor glycoproteins Ib/IX and IIb/IIIa. Although substantial progress has been made in defining vWF structure-function relationships, there are conflicting data regarding the location of its collagen-binding site(s). Possible collagen-binding sites have been localized in the A1 and A3 domains of vWF. To study the proposed binding sites, we have expressed cDNA sequences encoding the A1 and A3 domains of vWF in Escherichia coli and purified the resulting proteins from bacterial inclusion bodies. In addition, a chimeric molecule containing residues 465-598 of the vWF A1 domain polypeptide (vWF-A1) fused in frame to residues 1018-1114 of the vWF A3 domain polypeptide (vWF-A3) was also expressed. Each of the three recombinant proteins purified as a monomer and contained a single disulfide bond. As previously reported (Cruz, M. A., Handin, R. I., and Wise, R. J. (1993) J. Biol. Chem. 268, 21238-21245), recombinant vWF-A1 inhibited ristocetin-induced platelet agglutination, but did not compete with vWF multimers for collagen binding. In contrast, vWF-A3 inhibited the binding of multimeric vWF to immobilized collagen, but did not inhibit ristocetin-induced platelet agglutination. Metabolically labeled vWF-A3 bound to immobilized collagen in a saturable and reversible manner with a Kd of 1.8 x 10(-6) M. The vWF-A1/A3 chimera was bifunctional. It inhibited vWF binding to platelet glycoprotein Ib/IX with an IC50 of 0.6 x 10(-6) M and inhibited vWF binding to collagen with an IC50 of 0.5-1.0 x 10(-6) M. These results, taken together, provide firm evidence that the major collagen-binding site in vWF resides in the A3 domain.

Glycoprotein Ib can mediate endothelial cell attachment to a von Willebrand factor substratum.

Introduction of single amino acid substitutions into the C-terminal Arg-Gly-Asp-Ser (RGDS) site of von Willebrand Factor, referred to as RGD mutant vWF, selectively abrogated vWF binding to platelet glycoprotein IIb/IIIa (GpIIb/IIIa alpha IIb beta 3) and abolished human umbilical vein endothelial cell (HUVEC) spreading, but not attachment, to RGD mutant vWF (Beacham, D.A., Wise, R.J., Turci, S.M. and Handin, R. I. 1992. J. Biol. Chem. 167, 3409-3415). These results suggested that in addition to the vitronectin receptor (VNR, alpha V beta 3), a second endothelial membrane glycoprotein can mediate HUVEC adhesion to vWF. HUVEC attachment to wild-type (WT) and RGD-mutant vWF was reduced by two proteins known to block the vWF-platelet glycoprotein Ib/IX (GpIb/IX) interaction, the monoclonal antibody AS-7 and the recombinant polypeptide, vWF-A1. The addition of cytochalasin B or DNase I to disrupt potential GPIb alpha-cytoskeletal interactions enhanced the immunoprecipitation of endothelial GPIB alpha, caused HUVEC to round up, and increased HUVEC adhesion to RGD mutant vWF. These results indicate that while the VNR is the primary adhesion receptor for vWF, endothelial GPIb alpha can mediate HUVEC attachment to vWF. GpIb-dependent attachment could contribute to HUVEC adhesion under conditions when cell surface expression of the VNR is downregulated, and VNR-dependent adhesion is reduced.

The interaction of the von Willebrand factor-A1 domain with platelet glycoprotein Ib/IX. The role of glycosylation and disulfide bonding in a monomeric recombinant A1 domain protein.

The interaction of von Willebrand factor (vWF) with platelet glycoprotein Ib/IX plays an important role in primary hemostasis. Previous studies have localized the GpIb alpha binding domain of vWF to amino acid residues 449-728, a region containing the vWF-A1 domain. In order to assess the role of A1 domain structure in vWF binding functions, a cDNA encoding residues 475-709 of vWF was expressed in Escherichia coli (non-glycosylated) and in Chinese hamster ovary (CHO) cells (glycosylated). These recombinant proteins contain a single intrachain disulfide bond between C509 and C695 and were purified as monomers with apparent molecular weights of 36,000 (E. coli) and 39,000 (CHO). 35S-Labeled-vWF-A1 proteins bound directly to GpIb/IX receptors on platelets. The non-glycosylated form had a slightly higher affinity (Kd = 1.4 +/- 0.4 microM) than the glycosylated vWF-A1 protein (Kd = 4.5 +/- 0.9 microM) but had similar binding capacity of 28,000 GpIb/IX-specific binding sites per platelet. Additionally, both recombinant vWF-A1 proteins bound to heparin but neither bound to immobilized type I and III collagen. Both E. coli- and CHO-derived vWF-A1 proteins inhibited ristocetin-induced platelet agglutination with IC50 values of 300 and 700 nM, respectively. Reduction of the only disulfide bond between C509 and C695 abolished platelet binding activity at concentration up to 2 microM of protein. Confirmation of the importance of the 509-695 disulfide bond was obtained from a full-length vWF mutant containing substitutions at C509 and C695 (C509/695S) which failed to bind to the platelet GpIb/IX receptor. These studies document that vWF-A1 domain can bind to GpIb/IX and heparin but not collagen, and that binding to GpIb/IX requires an intact disulfide bond between C509 and C695. Furthermore, glycosylation increases the solubility but reduces binding affinity of recombinant vWF A1.

Autosomal recessive transmission of hemophilia A due to a von Willebrand factor mutation.

The differential diagnosis of the genetic bleeding disorders, hemophilia A and von Willebrand disease, is occasionally confounded by the close molecular relationship of coagulation factor VIII and von Willebrand factor (vWF). This report describes the autosomal inheritance of a hemophilia A phenotype due to a mutation of vWF that results in defective factor VIII binding. The proband was a female patient with low levels of factor VIII activity. Polymerase chain reaction (PCR) amplification and DNA sequencing were employed to examine exons encoding the putative factor VIII binding domain of vWF. The patient was found to be homozygous for a single point mutation causing a Thr-->Met substitution at amino acid position 28 in the mature vWF subunit. The phenotypic expression of the mutation was determined to be recessive because heterozygous family members were clinically unaffected. Recombinant vWF containing the observed amino acid substitution was expressed in COS-1 cells. The mutant vWF was processed and secreted normally, and was functionally equivalent to wild-type vWF in its ability to bind to platelets. However, the mutant failed to bind factor VIII, demonstrating that the mutation was functionally related to the observed hemophilia phenotype. The family we describe demonstrates the recessive inheritance of a recently recognized class of genetic bleeding disorders, we call "autosomal hemophilia." We conclude that vWF mutation may be an under recognized cause of hemophilia, especially in cases where the inheritance pattern is not consistent with X-linked transmission.

Transient expression of recombinant glycoprotein Ib alpha polypeptides in COS cells that inhibit von Willebrand factor binding to the platelet glycoprotein Ib/IX complex.

The cDNA encoding the glycoprotein Ib alpha polypeptide has been expressed in COS cells. Transfection with full-length cDNA and a cDNA truncated at an internal XbaI site produced a recombinant polypeptide doublet with estimated molecular weights of 48 and 46 kDa (rGpIb alpha L318) which could be resolved into a single band of molecular weight 36 kDa following digestion with endoglycosidase F. A portion of the truncated polypeptide was retained in the endoplasmic reticulum of COS cells and slowly released. A second fraction was rapidly secreted into COS cell-conditioned medium and could be used for functional studies. Soluble rGpIb alpha L318 harvested from COS cell-conditioned medium inhibited ristocetin-dependent binding of [125I]-vWF to fixed washed human platelets (IC50 20 nM). Binding was inhibited by reduction and alkylation of "rGp1b alpha L318" suggesting the need for a critical disulfide bond to maintain biological activity of the recombinant polypeptide. We conclude that the recombinant polypeptides produced by transfection of GpIb alpha cDNA into heterologous cells are secreted, soluble, and can inhibit vWF binding to platelet GpIb/IX.

Selective inactivation of the Arg-Gly-Asp-Ser (RGDS) binding site in von Willebrand factor by site-directed mutagenesis.

In order to assess the requirement for the Arg-Gly-Asp-Ser (RGDS) consensus adhesion sequence in von Willebrand factor (vWF) for vWF binding to platelets and endothelial cells, point mutations were introduced into this sequence by site-directed mutagenesis. A glycine to alanine substitution yielded RADS-vWF, while an aspartate to glutamate substitution resulted in RGES-vWF. Recombinant RADS-vWF and RGES-vWF, purified from transformed Chinese hamster ovary cells, were compared with recombinant wild type vWF (WT-vWF) in functional assays with platelets and human umbilical vein endothelial cells (HU-VECs). High molecular weight RADS-vWF and RGES-vWF multimers inhibited binding of 125I-vWF to a mixture of insolubilized native type I and III collagen and competed effectively with 125I-vWF for binding to formalin-fixed platelets in the presence of ristocetin, indicating functional collagen and platelet glycoprotein Ib binding. However, RADS-vWF and RGES-vWF were unable to displace the binding of 125I-vWF to thrombin or ADP-activated platelets. The attachment of HUVECs to either RADS-vWF or RGES-vWF coated surfaces was reduced and spreading was almost completely inhibited, compared with WT-vWF. We conclude that point mutations of the RGDS sequence in vWF selectively impair binding to platelet glycoprotein IIb/IIIa and the HUVEC vitronectin receptor.

Functional analysis of a recombinant glycoprotein Ib alpha polypeptide which inhibits von Willebrand factor binding to the platelet glycoprotein Ib-IX complex and to collagen.

By deletion mutagenesis and transient expression in COS cells, a 96-amino acid hydrophilic sequence in the glycoprotein Ib alpha polypeptide located between L220 and L318 was identified which appeared to contain its von Willebrand factor- (vWF) binding site. The cDNA encoding this fragment was then expressed in Escherichia coli and purified from the bacterial cell lysate. The recombinant polypeptide, rGpIb alpha Q221-L318, was monomeric and had an apparent molecular weight of 14,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It inhibited both ristocetin-induced binding of 125I-vWF to fixed washed platelets and ristocetin-induced platelet agglutination. The recombinant polypeptide also inhibited the binding of 125I-vWF to immobilized type I and III collagen. Inhibition of 125I-vWF binding to platelets and collagen was dose-dependent, with IC50 values of 500 and 200 nM rGpIb alpha Q221-L318, respectively. Fifty % inhibition of ristocetin-induced platelet agglutination required 500 nM rGpIb alpha Q221-L318. Although rGpIb alpha Q221-L318 inhibited vWF binding to collagen it did not, itself, bind to collagen-coated surfaces. Reduction of the disulfide bond between C248 and C264 abolished activity. 125I-rGpIb alpha Q221-L318 bound directly to GpIb/IX sites on multimeric vWF. These studies document that a portion of the sequence between Q221 and L318 is needed for recognition and binding to vWF and that binding requires an intact disulfide bond between C248 and C264. The binding of this recombinant polypeptide to vWF multimers inhibits vWF interaction with two important substrates, platelet GpIb/IX and collagen.

von Willebrand factor binding to platelet GpIb initiates signals for platelet activation.

The hypothesis that von Willebrand factor (vWF) binding to platelet membrane glycoprotein Ib (GpIb) initiates intracellular pathways of platelet activation was studied. We measured the biochemical responses of intact human platelets treated with ristocetin plus vWF multimers purified from human cryoprecipitate. vWF plus ristocetin causes the breakdown of phosphatidylinositol 4,5-bisphosphate, the production of phosphatidic acid (PA), the activation of protein kinase C (PKC), increase of ionized cytoplasmic calcium ([Ca2+]i), and the synthesis of thromboxane A2. PA production, PKC activation, and the rise of [Ca2+]i stimulated by the ristocetin-induced binding of vWF multimers to platelets are inhibited by an anti-GpIb monoclonal antibody, but are unaffected by anti-GpIIb-IIIa monoclonal antibodies. Indomethacin also inhibits these responses without impairing platelet aggregation induced by vWF plus ristocetin. These results indicate that vWF binding to platelets initiates specific intraplatelet signaling pathways. The mechanism by which this occurs involves an arachidonic acid metabolite-dependent activation of phospholipase C after vWF binding to platelet membrane GpIb. This signal then causes PKC activation and increases of [Ca2+]i, which promote platelet secretion and potentiate aggregation.

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.

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.