Mapping the platelet proteome: a report of the ISTH Platelet Physiology Subcommittee.

Proteomic technology has the potential to transform the way we analyze platelet biology, through the determination of platelet protein composition and its modification upon stimulation and with disease. We are a considerable way from achieving these goals, however, because of significant limitations in current methodology. It is therefore important to consider the extent to which these aims can be met and the way that proteomic data should be presented and used. These issues are discussed in the present paper by the Platelet Physiology Subcommittee of the ISTH Scientific Standardisation Committee (SSC). It is recommended that proteomic information be combined with data from other experimental approaches to establish a database on protein expression and function in platelets.

Adeno-associated virus (AAV) Rep protein enhances the generation of a recombinant mini-adenovirus (Ad) utilizing an Ad/AAV hybrid virus.

Mini-adenoviruses (mAd) deleted of all viral coding regions represent an emerging approach for transgene expression. We have exploited the unique features of the adeno-associated virus (AAV) terminal repeats within the context of an adenovirus-adeno-associated hybrid virus (Ad/AAV) as a strategy for rapid and efficient generation of mAd. Excision and generation of mAd from the parental Ad/AAV hybrid vector was achieved in 293 cells through recombination but without selection for mAd production. Analysis of mAd isolated from 293 cells indicated that mAd DNA exists as monomer and dimer forms within the recombinant viral capsid. Formation of recombinant mAd was significantly increased using an AAV Rep78- or Rep68-expressing cell line through Rep-mediated excision utilizing the AAV terminal repeat sequences present in the Ad/AAV hybrid virus genome. The mAd viruses were infectious and able to transfer functional gene to A549 and HeLa cells. This approach is rapid and efficient, thereby providing a simplified methodology for generating mAd with functional transducing capabilities.

The propeptide domain of membrane type 1-matrix metalloproteinase acts as an intramolecular chaperone when expressed in trans with the mature sequence in COS-1 cells.

It has been assumed that cleavage of the N-terminal propeptide domain of membrane type-1 matrix metalloproteinase (MT1-MMP) is required for enzyme function. We recently demonstrated that the propeptide domain of MT1-MMP is not cleaved and actually is required for function of the membrane-bound enzyme in transfected COS-1 cells (Cao, J., Drews, M., Lee, H. M., Conner, C., Bahou, W. F., and Zucker, S. (1998) J. Biol. Chem. 273, 34745-34752). In this report, we have inserted the cDNA encoding the signal and propeptide sequences of MT1-MMP (MT(1-109)) and the cDNA encoding propeptide-deleted mature MT1-MMP (MT delta pro) in expression vectors that were then transfected into matrix metalloproteinase-deficient COS-1 cells. Co-expression of both the mature sequence and the prosequence of MT1-MMP as independent polypeptides (in trans) in COS-1 cells resulted in reconstitution of MT1-MMP function in terms of facilitating (125)I-labeled tissue inhibitor of metalloproteinase 2 binding to transfected cells and subsequent activation of progelatinase A. Transfection of cells with either cDNA alone resulted in non-functional cells. These results are consistent with the propeptide sequence of MT1-MMP functioning as an intramolecular chaperone involved in protein folding and trafficking to the cell surface.

Platelet glycoprotein Ib: a zinc-dependent binding protein for the heavy chain of high-molecular-weight kininogen.

Domains 3 and 5 of high-molecular-weight kininogen (HK) have been shown to bind to platelets in a zinc-dependent reaction. However, the platelet-binding proteins responsible for this interaction have not been identified. We have focused on the platelet-binding site for the heavy chain (domain 3), which we approached using a domain 3-derived peptide ligand and isolated binding proteins by affinity chromatography. The domain 3-derived peptide, thrombin, HK, factor XII, as well as antibody to glycocalicin (the N-terminal portion of the alpha chain of GPIb) recognized a protein at 74 kD. We also isolated the thrombin receptor (PAR 1) at 45 kD, however, none of the above-mentioned ligands bound to this protein. Isolation of platelet membrane proteins using a monoclonal anti-glycocalicin antibody column revealed the same HK binding protein at 74 kD, which was reactive with anti-GPIb and represents a GPIb fragment. By photoaffinity labeling, HK interacted with membrane GPIb, which was then isolated in native form (135 kD) along with gC1qR, a ligand for the HK light chain. Finally, (125)I-HK binding to platelets was significantly inhibited by the anti-GPIb antibody. These results suggest that the GPIb alpha chain, a known thrombin binding protein, is also one of the zinc-dependent platelet membrane binding sites for HK domain 3.

Proteolytically activated receptor-3. A member of an emerging gene family of protease receptors expressed on vascular endothelial cells and platelets.

Macromolecular assembly and generation of serine proteases on cellular surfaces is critically involved in regulation of hemostatic, inflammatory, or fibrinolytic pathways. The concept that a number of these serine proteases may effect cellular activation and proliferative responses has engendered an emerging paradigm focusing on the molecular mechanisms regulating cellular/protease interactions. Previous data suggest that some of these cellular responses are mediated by a novel class of G protein-coupled proteolytically activated receptors. Proteolytically activated receptor-3 (PAR-3) is the third member of this rapidly emerging gene family, all three of which (PAR-1, PAR-2, PAR-3) are known to co-cluster in the human genome, and are expressed on vascular endothelial cells, cells which critically regulate the hemostatic repertoire. This review will focus on the genetics of these receptors (emphasizing recent advances in the identification and characterization of PAR-3), review known structure/function similarities, and outline potential links in regulation of the hemostatic response by protease generation on the endothelial cell surface.

Human factor VIII can be packaged and functionally expressed in an adeno-associated virus background: applicability to haemophilia A gene therapy.

Adeno-associated virus (AAV) is a single-stranded DNA parvovirus displaying several attractive features applicable to haemophilia A gene therapy, including nonpathogenicity and potential for long-term transgene expression from either integrated or episomal forms. We have generated and characterized two B-domain-deleted (BDD) fVIII mutants, deleted in residues Phe756 to Ile1679 (fVIIIdelta756-1679) or Thr761 to Asn1639 (fVIIIdelta761-1639). [35S]metabolic labelling experiments and immunoprecipitation demonstrated intact BDD-fVIII of the predicted size in both lysates and supernatants (Mr approximately 155 kD for fVIIIdelta756-1679 and Mr approximately 160 kD for fVIIIdelta761-1639) after transient transfection into COS-1 cells. Functional fVIII quantification appeared maximal using fVIIIdelta761-1639, as evaluated by Coatest and clotting assay (98+/-20mU/ml/1x10(6) cells and 118+/-29 mU/ml/1x10(6) respectively, collection period 48 h). To bypass potential size limitations of rAAV/fVIII vectors, we expressed fVIIIdelta761-1639 using a minimal human 243 bp cellular small nuclear RNA (pHU1-1) promoter, and demonstrated VIII activity approximately 30% of that seen using CMV promoter. This BDD-fVIII (rAAV(pHU1-1) fVIIIdelta761-1639) can be efficiently encapsidated into rAAV (107% of wild type), as demonstrated by replication centre and DNAase sensitivity assays. A concentrated recombinant viral stock resulted in readily detectable factor VIII expression in COS-1 cells using a maximally-achievable MOI approximately 35 (Coatest 15 mU/ml; clotting assay 25+/-20 mU/ml/1x10(6) cells). These data provide the first evidence that rAAV is an adaptable virus for fVIII delivery, and given the recent progress using this virus for factor IX delivery in vivo, provide a new approach towards definitive treatment of haemophilia A.

The propeptide domain of membrane type 1 matrix metalloproteinase is required for binding of tissue inhibitor of metalloproteinases and for activation of pro-gelatinase A.

Activation of secreted latent matrix metalloproteinases (MMPs) is accompanied by cleavage of the N-terminal propeptide, thereby liberating the active zinc from binding to the conserved cysteine in the pro-domain. It has been assumed that an analogous mechanism is responsible for the activation of membrane type 1 MMP (MT1-MMP). Using recombinant wild-type MT1-MMP cDNA and mutant cDNAs transfected into COS-1 cells lacking endogenous MT1-MMP, we have examined the function of the propeptide domain of MT1-MMP. MT1-MMP was characterized by immunoblotting, surface biotinylation, gelatin substrate zymography, and 125I-tissue inhibitor of metalloproteinases 2 (TIMP-2) binding. In contrast to wild-type MT1-MMP-transfected COS-1 cells, transfected COS-1 cells containing a deletion of the N-terminal propeptide domain of MT1-MMP or a chimeric construction (substitution of the pro-domain of MT1-MMP with that of collagenase 3) were functionally inactive in terms of binding of 125I-labeled TIMP-2 to the cell surface and initiating the activation of pro-gelatinase A. These results support the concept that in its native plasma membrane-inserted form, the pro-domain of MT1-MMP plays an essential role in TIMP-2 binding and subsequent activation of pro-gelatinase A.

The human proteinase-activated receptor-3 (PAR-3) gene. Identification within a Par gene cluster and characterization in vascular endothelial cells and platelets.

Proteolytically activated receptors (PARs) represent an emerging subset of seven transmembrane G protein-coupled receptors that mediate cell activation events by receptor cleavage at distinct scissile bonds located within receptor amino termini. Differential genomic blotting using a yeast artificial chromosome known to contain the PAR-1 and PAR-2 genes identified the PAR-3 gene within a PAR gene cluster spanning approximately 100 kilobases at 5q13. The PAR-3 gene is relatively small (approximately 12 kilobases); and, like the PAR-1 and PAR-2 genes, it displays a two-exon structure, with the majority of the coding sequence and the proteolytic cleavage site contained within the larger second exon. Sequence analysis of the 5'-flanking region demonstrates that the promoter is TATA-less, similar to that seen with PAR-1, with the identification of nucleic acid motifs potentially involved in transcriptional gene regulation, including AP-1, GATA, and octameric sequences. PAR-3 transcripts were apparent in human vascular endothelial cells, although at considerably lower levels than those of PAR-1 and not significantly modulated by the endothelial cell stimulus tumor necrosis factor-alpha. Likewise, although PAR-3 mRNA was evident in human platelets, receptor cell surface expression was modest (approximately 10%) compared with that of PAR-1. Thus, although PAR-3 is postulated to represent a second thrombin receptor, its modest endothelial cell and platelet expression suggest that PAR-3 activation by alpha-thrombin is less relevant for physiological responses in these mature cells. Rather, given its disparately greater expression in megakaryocytes (and megakaryocyte-like human erythroleukemia cells), a regulatory role in cellular development (by protease activation) could be postulated.

Vascular endothelial growth factor induces tissue factor and matrix metalloproteinase production in endothelial cells: conversion of prothrombin to thrombin results in progelatinase A activation and cell proliferation.

Production of vascular endothelial growth factor (VEGF) by cancer cells at invasive and metastatic sites is an important aspect of tumor angiogenesis. Although known primarily as a mitogen and a vascular permeability factor (VPF) for endothelial cells, VEGF/VPF has been proposed to induce the expression of procoagulant factors in endothelial cells. In this study, we have explored the ramifications of VEGF induction of tissue factor (TF) in human umbilical vein endothelial cells (HUVECs) and subsequent activation of progelatinase A. Within 3 hr of incubation with VEGF/VPF, endothelial cells accelerate TF generation as measured using chromogenic substrate assays for coagulation factors Xa and thrombin. Incubation of VEGF/VPF-pre-treated cells with prothrombin and factors X, Va, and VIIa at 37 degrees C and subsequent generation of thrombin resulted in activation of secreted endothelial progelatinase A as demonstrated by gelatin zymography. Anti-thrombin III or antibodies to TF inhibited thrombin generation and progelatinase A activation. VEGF/VPF also directly increased HUVEC secretion of interstitial collagenase, tissue inhibitor of metalloproteinases (TIMP-1) and, to a lesser extent, gelatinase A. The effect of thrombin on endothelial proliferation in serum-free media was examined. Thrombin was a growth factor for HUVECs at a lower dose than that required for progelatinase A activation. Whereas TIMP-2 abrogated thrombin-induced progelatinase A activation, it had no significant effect on thrombin-induced endothelial cell growth. We propose that an early step in tumor angiogenesis involves VEGF-induced thrombin generation and increased MMP production with subsequent activation of endothelial progelatinase A and degradation of the underlying basement membrane.

Tissue inhibitor of metalloproteinase-2 (TIMP-2) binds to the catalytic domain of the cell surface receptor, membrane type 1-matrix metalloproteinase 1 (MT1-MMP).

It has been proposed that tissue inhibitor of metalloproteinase-2 (TIMP-2), in stoichiometric concentrations, serves as an intermediate in progelatinase A activation by binding to activated membrane type 1-matrix metalloproteinase 1 (MT1-MMP) on the plasma membrane. An MT1-MMP-independent cell surface receptor for TIMP-2 has also been postulated. To clarify TIMP-2 binding, we have performed 125I-TIMP-2 binding studies on transfected COS-1 cells and endothelial cells. Specific receptors for TIMP-2 were identified on COS-1 cells transfected with MT1-MMP cDNA, but not on vector-transfected cells. Treatment of MT1-MMP transfected COS-1 cells with a hydroxamic acid inhibitor of MMPs, CT-1746, but not an inactive stereoisomer, CT-1915, produced dose-dependent inhibition of specific TIMP-2 binding comparable with that noted with excess unlabeled TIMP-2. This result suggests that TIMP-2 binds to the zinc catalytic site of MT1-MMP. As demonstrated by the limited competition for binding of C-terminal deleted TIMP-2, the C-terminal domain of TIMP-2 participates in binding to MT1-MMP. Cross-linking studies followed by immunoprecipitation using antibodies to MT1-MMP were employed to identify 125I-TIMP-2.MT1-MMP complexes in MT1-MMP-transfected COS-1 cell membrane extracts. TIMP-2 receptors were also identified on concanavalin A-treated human umbilical vein endothelial cells; inhibition of TIMP-2 binding with CT-1746 was demonstrated.

Characterization of the initial alpha-thrombin interaction with glycoprotein Ib alpha in relation to platelet activation.

We have evaluated the properties of alpha-thrombin interaction with platelets within 1 min from exposure to the agonist, a time frame during which most induced activation responses are initiated and completed. Binding at 37 degrees C was rapidly reversible and completely blocked by a monoclonal antibody, LJ-Ib10, previously shown to be directed against the alpha-thrombin interaction site on glycoprotein (GP) Ib alpha. By 2-5 min, however, binding was no longer fully reversible and was only partially inhibited by the anti-GP Ib alpha antibody. Results were similar at room temperature (22-25 degrees C), whereas the initial characteristics of alpha-thrombin interaction with platelets were preserved for at least 20 min at 4 degrees C. Equilibrium binding isotherms obtained at the latter temperature were compatible with a two-site model, but the component ascribed to GP Ib alpha, completely inhibited by LJ-Ib10, had "moderate" affinity (kd on the order of 10(-8) M) and relatively high capacity, rather than "high" affinity (kd on the order of 10(-10) M) and low capacity as currently thought. The parameters of alpha-thrombin binding to intact GP Ib alpha on platelets at 4 degrees C corresponded closely to those measured with isolated GP Ib alpha fragments regardless of temperature. Blocking the alpha-thrombin-GP Ib alpha interaction caused partial inhibition of ATP release and prevented the association with platelets of measurable proteolytic activity. These results support the concept that GP Ib alpha contributes to the thrombogenic potential of alpha-thrombin.

The human thrombin receptor gene and the 5q-syndrome.

The human thrombin receptor gene has been localized to band q13 of chromosome 5, a site that is at or contiguous to the common proximal breakpoint found in the majority of patients with interstitial deletions involving 5q (5q- syndrome; refractory anemia with dysmegakaryocytopoiesis). Recent evidence suggests that the thrombin receptor may represent the prototype of an emerging family of proteolytically activated receptors that may be clustered within this region of the human genome. The phenotypic heterogeneity evident in patients with the 5q- syndrome may be explained by two (or more) distinct molecular defects-one associated with megakaryocytic dyspoiesis and the other dysregulated myeloid growth potentially related to development of leukemogenesis. Because the thrombin receptor is known to mediate proliferative effects on diverse cells including vascular smooth muscle cells, endothelial cells and megakaryocytes, we have studied the role of this receptor in the pathogenesis of this syndrome using fluorescent in situ hybridization (FISH) analysis. Dual-label FISH using a q12-specific genomic fragment and the TR gene was completed using interphase and metaphase analysis from seven patients with a del(5)(q13q33). These data unequivocally demonstrate that the thrombin receptor gene is located centromeric to the common proximal breakpoint, and is grossly present in the seven patients containing this specific interstitial deletion. Additionally, one patient demonstrated a small proximal rearrangement, most likely representing a paracentric inversion, which has not previously been described within the intact region centromeric to the breakpoint. The biological properties of proteolytically activated receptors are reviewed in more detail, with a focus on the molecular genetics of the thrombin receptor and its potential role in megakaryoctyopoiesis.

Mitogenic responses mediated through the proteinase-activated receptor-2 are induced by expressed forms of mast cell alpha- or beta-tryptases.

The proteinase-activated receptor-2 (PAR-2) is the second member of a putative larger class of proteolytically activated receptors that mediate cell activation events by receptor cleavage or synthetic peptidomimetics corresponding to the newly generated N-terminus. To further study the previously identified mitogenic effects of PAR-2, we used the interleukin-3 (IL-3)-dependent murine lymphoid cell line, BaF3, for generation of stable cell lines expressing PAR-2 (BaF3/PAR-2) or the noncleavable PAR-2 mutant PAR-2(Arg36 --> Ala36). Only BaF3 cells expressing either wild-type or mutated receptor exhibited mitogenic responses when grown in IL-3-deficient media supplemented with PAR-2 activating peptide (SLIGRL, PAR39-44). This effect was dose dependent with an EC50 of approximately 80 micromol/L, sustained at 24, 48, and 72 hours, and was also demonstrable using thrombin receptor peptide TR42-47. Because tryptase shares approximately 70% homology with trypsin (previously shown to activate PAR-2), we studied recombinantly expressed forms of alpha- and beta-tryptases as candidate protease agonists for PAR-2. Hydrolytic activity of the chromogenic substrate tosyl-glycyl-prolyl-argly-4-nitroanilide acetate was present as a sharp peak at Mr approximately 130, confirming the presence of secretable and functionally active homotetrameric alpha- and beta-tryptases in transfected COS-1 cells. Dose-dependent proliferative responses were evident using either secreted form of tryptase with maximal responses seen at approximately 3 pmol/L (0.1 U/L). Receptor proteolysis was necessary and sufficient for mitogenesis because active site-blocked tryptase failed to induce this response, and proliferative responses were abrogated in BaF3 cells expressing PAR-2(Arg36 --> Ala36). These results specifically identify both forms of mast cell tryptases as serine protease agonists for PAR-2 and have implications for elucidating molecular mechanisms regulating cellular activation events mediated by proteases generated during inflammatory, fibrinolytic, or hemostatic-regulated pathways.

The human thrombin receptor and proteinase activated receptor-2 genes are tightly linked on chromosome 5q13.

The thrombin receptor (TR) and proteinase activated receptor-2 (PAR-2) may represent the prototypes of an emerging family of cell-surface receptors that effect cell activation events mediated by serine proteases generated during inflammatory, fibrinolytic or haemostatic-regulated pathways. To further characterize the molecular genetics of these receptors, we have refined the genetic and physical mapping of both PAR-2 and TR. Utilization of two distinct radiation hybrid mapping panels with different levels of resolution demonstrated that both genes are tightly linked to the microsatellite markers D5S424, D5S1977, D5S2529 and D5S2596 (in order of decreasing LOD scores, from 13.7 for D5S424 to 7.7 for D5S2596). Physical mapping using yeast artificial chromosomes (YACs) and inversion field gel electrophoresis demonstrated that they are maximally separate by 90 kb. If the association of TR and PAR-2 genes resulted from a relatively recent gene duplication event from a common ancestral gene, these observations provide a general framework for the identification of gene transcripts representing alternative proteolytically activated receptors which may be clustered within this region of the human genome. These observations are especially relevant given recent evidence that murine and human platelets express alternative signalling mechanisms or receptors for thrombin.

In vivo gene transfer into rat arterial walls with novel adeno-associated virus vectors.

PURPOSE: We studied the ability of recombinant adeno-associated virus (rAAV) vectors to achieve gene transfer in vivo to intact rat carotid arteries. METHODS: Isolated segments of uninjured rat carotid arteries were incubated with (1) rAAV vectors that expressed a beta-galactosidase gene, (2) a related vector with no promoter, or (3) a normal saline solution. Gene transfer was evaluated with in situ polymerase chain reaction (PCR). Transgene expression was assessed at intervals that ranged from 24 hours to 2 months by measurement of beta-galactosidase activity and protein mass in tissue extracts with fluorometric and enzyme-linked immunosorbent assays, respectively. Dose dependence of expression was determined for virus concentrations that ranged from 5 x 10(4) to 5 x 10(5) infectious units (iu)/ml. RESULTS: Light microscopic analysis of in situ PCR-stained histologic sections of transduced vessel walls showed approximately 90% of intimal and medial cell nuclei contained the beta-galactosidase gene, compared with none in control arteries. In vivo beta-galactosidase expression was (1) highest 24 hours after gene transfer, (2) elevated for 1 month, and (3) dose responsive. CONCLUSIONS: rAAV vectors can mediate focal gene transfer into the intact rat carotid artery with detectable levels of transgene expression for 1 month and are potentially useful agents for in vivo gene transfer into intact arteries.

Characterization of recombinant adeno-associated virus-2 as a vehicle for gene delivery and expression into vascular cells.

BACKGROUND: We have used wild-type and recombinant adeno-associated virus-2 (AAV) to study transduction, replication efficiencies, functional protein expression, and gene delivery to vascular cells in vitro and in vivo. METHODS: Recombinant adeno-associated virus-2 (rAAV) plasmids (ranging in size to 110% of wild-type AAV) driven by 6 distinct promoters upstream of a beta-galactosidase cassette were effectively used for generation of replication-deficient virus, with titers consistently ranging from 2.5 x 10(5) IU/mL. AAV infectivity and replication in human umbilical vein endothelial cells (HUVEC) were unrelated to cellular proliferative index establishing the potential utility of the virus for transduction of quiescent vascular cells. Long-term cultures of AAV-infected HUVEC established the presence of episomal forms at 18 days, although chromosome 19-specific integration was not evident. Functional beta-galactosidase activity approximately 400% above control was evident in HUVEC using either a murine collagen alpha 1(I) promoter (pTRCol alpha 1(I) beta) or CMV promoter (pTRCMV beta). RESULTS: Based on these initial data, in vivo studies were completed using a rat carotid artery model. Both wild-type AAV (titers -1X10(9) IU/mL) and rAAV (pTRCol alpha 1(I) beta or pTRCMV beta) efficiently infected vascular cells in vivo with endothelial and vascular smooth muscle cell transduction frequencies approaching 90% as judged by DNA in situ polymerase chain reaction, with no evidence for disrupted vessel architecture. Protein expression using total vessel extracts at 48 hours postinfection demonstrated 20-fold increase in functional beta-galactosidase activity using pTRCol alpha 1(I) beta compared to saline-injected controls vessels (799 +/- 236 microU/mg protein vs 40.7 +/- 17 microU/mg protein). CONCLUSIONS: These data provide the first evidence that rAAV may be adapted for directed high-level transgene delivery and expression into normally quiescent vascular endothelial and smooth muscle cells both in vitro and in vivo.

Permissive role of nitric oxide in endothelin-induced migration of endothelial cells.

Endothelin (ET) synthesis is enhanced at sites of ischemia or in injured vessels. The purpose of this study was to explore the possibility of autocrine stimulation of endothelial cell migration by members of the endothelin family. Experiments with microvascular endothelial cell transmigration in a Boyden chemotactic apparatus showed that endothelins 1 and 3, as well as a selective agonist of ETB receptor IRL-1620, equipotently stimulated migration. Endothelial cell migration was unaffected by the blockade of ETA receptor, but it was inhibited by ETB receptor antagonism. Based on our previous demonstration of signaling from the occupied ETB receptor to constitutive nitric oxide (NO) synthase (Tsukahara, H., Ende, H., Magazine, H. I., Bahou, W. F., and Goligorsky, M. S. (1994) J. Biol. Chem. 269, 21778-21785), we next examined the contribution of ET-stimulated NO production to endothelial cell migration. In three independent cellular systems, 1) migration and wound healing by microvascular endothelial cells, 2) wound healing by Chinese hamster ovary cells stably expressing ETB receptor with or without endothelial NO synthase, and 3) application of antisense oligodeoxynucleotides targeting endothelial NO synthase in human umbilical vein endothelial cells, an absolute requirement for the functional NO synthase in cell migration has been demonstrated. These findings establish the permissive role of NO synthesis in endothelin-stimulated migration of endothelial cells.