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PubMed2000

Blood platelet activation evaluated by flow cytometry: optimised methods for clinical studies.

A variety of flow cytometry techniques are in use to evaluate in vivo blood platelet activation. We have in this study further developed and optimised these methods to be suitable for use in clinical studies. By preloading the Monovette EDTA vacuum blood sampling tubes with 1/8 vol 4% (w/v) paraformaldehyde (PFA), we were able to assess platelet CD62P (P-selectin) expression in whole blood with less than 0.2% activated platelets. No washing or neutralising steps were required to remove excess fixative. Both basal and agonist-stimulated CD62P expression were stable for at least 48 h after sampling. The standard curve was linear from 1.9 (basal) to 8.1 x 10(3) (TRAP-stimulated) molecules of equivalent soluble fluorochrome units (MESF) in phycoerythrein-conjugated anti-CD62P labelled whole blood samples. These assay conditions were also well suited for assessment of platelet expression of CD41, CD42a, CD61 and CD63. The preanalytic storage period was extended from 10 min to at least 2 h for platelet PAC-1 and fibrinogen binding analysis by preloading Monovette citrate tubes with 8/10 vol buffer. With PFA preloading, blood sampled into citrated tubes could be analysed for fractions of microparticles and platelet-platelet aggregates as well as for aggregate size.

Adult↗
PubMed2002

Altered megakaryocyte-platelet-haemostatic axis in patients with acute stroke.

Platelet function is accentuated in acute ischaemic stroke (IS) and may also be altered in haemorrhagic stroke. Whether these changes are a direct reaction to the stroke or are secondary to changes in megakaryocytes (MKs) is unknown. To determine whether MKs are altered in acute stroke we studied 24 patients (18 with ischaemic stroke, six with haemorrhagic stroke) within 3 days of symptom onset, and 14 matched controls. MK ploidy (DNA content, N), size (forward scatter, FSC, arbitrary units), granularity (side scatter, SSC, arbitrary units) and glycoprotein (GP) IIIa expression (arbitrary units) were assessed by flow cytometry. Platelet size (MPV, fl), platelet count (PC, x109/l), circulating reticulated platelets (%), and cutaneous bleeding time (s) were also measured. MK ploidy 22.5 (2.7) vs. 20.6 (1.7) (2p = 0.014); FSC 629 (51) vs. 594 (41) (2p = 0.025); and SSC 843 (88) vs. 776 (76) (2p = 0.020) were each increased, whereas bleeding time 318 (102) vs. 401 (94) (2p = 0.050) was decreased in patients with acute stroke as compared with controls. Trends to increased MK GP IIIa expression and reticulated platelets were also apparent. In a post hoc analysis, the increase in MK ploidy was most prominent in patients with a prior history of hypertension. Ischaemic stroke was associated with non-significant increases in MK ploidy, size, and granularity. However, MK parameters were different in acute haemorrhagic stroke as compared with controls: MK ploidy 23.0 (1.8) vs. 20.6 (1.7) (2p = 0.018); MK FSC 637 (21) vs. 594 (41) (2p = 0.050); MK SSC 872 (41) vs. 776 (76) (2p = 0.020), changes which could be related to the high prevalence of hypertension (83%) in this group. These results demonstrate that pro-thrombotic changes in the megakaryocyte-platelet-haemostatic axis (MPHA) are present in acute stroke. Although megakaryocyte changes are likely, in part, to be secondary to the stroke, they could also precede the stroke and therefore explain some of the increased platelet function observed in acute stroke.

Aged↗
PubMed2002

Flow cytometric assay of platelet glycoprotein receptor numbers in hypercholesterolemia.

In this study, platelet glycoprotein (Gp) receptor numbers were measured by a flow cytometric assay using Cytoquant Gp in seven hypercholesterolemic and five normal subjects. Thrombin receptor agonist peptide (TRAP) was used to activate platelets. In hypercholesterolemia the Gp receptor numbers per resting platelet were found to be: 38,629 +/- 8,538 (GpIIb/IIIa), 22,269 +/- 5,628 (GpIb), 37,037 +/- 9,810 (GpIIIa), 224 +/- 504 (CD62-P). After activation, receptor numbers were determined to be: 56,399 +/- 9,003 (GpIIb/IIIa), 10,970 +/- 5,319 (GpIb), 50,715 +/- 7,904 (GpIIIa), 1,222 +/- 687 (P-selectin). In the normal group before the activation, receptor numbers were: 43,828 +/- 8,862 (GpIIb/IIIa), 22,166 +/- 3,847 (GpIb), 42,351 +/- 1,049 (GpIIIa), 62 +/- 139 (CD62-P), After activation, receptor numbers were determined to be: 60,573 +/- 4,294 (GpIIb/IIIa), 13,003 +/- 4,118 (GpIb), 52, 067 +/- 1,039 (GpIIIa), 3,608 +/- 1,508 (CD62-P). In hypercholesterolemic subjects, GpIIb/IIIa and GpIIIa receptor numbers on activated platelets increased significantly, whereas P-selectin numbers remained unchanged. However, the GpIb levels decreased significantly. In the control group, after activation, GpIIb/IIIa and P-selectin receptors increased significantly, GpIIIa receptor numbers did not change significantly, whereas GpIb receptor numbers decreased significantly. When the GpIIb/IIIa, GpIb, GpIIIa receptor numbers of the control group and hypercholesterolemic group were compared before and after activation, no significant changes were observed (P > 0.05). But P-selectin receptor numbers were significantly decreased in hypercholesterolemic patients compared to normals following TRAP activation (P < 0.05). In this study, the effect of hypercholesterolemia on platelet function was observed. The striking observation about present study was the marked decrease in P-selectin expression after activation in the hypercholesterolemics compared to normals. This finding suggests some sort of platelet dysfunction in these individuals.

Adult↗
PubMed2006

Aspirin resistance in coronary artery disease is correlated to elevated markers for oxidative stress but not to the expression of cyclooxygenase (COX) 1/2, a novel COX-1 polymorphism or the PlA(1/2) polymorphism.

Aspirin resistance (AR) is estimated to be present in 5-75% of patients and is related to increased cardiovascular mortality. However, the underlying mechanisms are mostly unknown. In the present study, AR was detected in 14 out of 55 patients (25%) with coronary artery disease. The presence of concomitant anti-inflammatory drugs did not affect AR. Plasma levels of thromboxane B(2) as well as the markers for oxidative stress and known platelet activators 8-isoprostane and lipid peroxidation products were significantly higher in aspirin-resistant individuals (349.3 pg/ml, 53.9 pg/ml, and 538 micromol/l) compared to controls (113.7 pg/ml, 10.3 pg/ml, and 32.2 micromol/l; P < 0.05, respectively). Platelet cyclooxygenase-1 (COX-1) and COX-2 mRNA and protein expression were without significant differences between the two groups. DNA sequencing detected a novel platelet COX-1 single nucleotide polymorphism (SNP) resulting in amino acid exchange at position 8 (Arg8/Trp8). The wild-type as well as the heterozygous and homozygous SNP were present in both patient groups without significant differences. The aspirin binding (Arg120) and acetylation site (Ser529) were unaffected in the samples tested. Neither was AR related to the platelet integrin PlA(1)/A(2) polymorphism. In conclusion, AR appears to be unrelated to differences in platelet COX-1 and COX-2 expression or to a novel platelet COX-1 SNP and the PlA(1)/A(2) SNP. However, a correlation exists to elevated eicosanoids generated by oxidative stress indicating COX-1-independent pathways for the generation of platelet activating molecules represent a potential cause for AR.

Antigens, Neoplasm↗
PubMed1998

In contrast to fibrinogen or fibrin, peptide and peptide mimetic binding to alphaIIbbeta3 (GPIIb-IIIa) does not cause outside-in signalling as judged by measurements of phosphatidylinositol 4,5-bisphosphate.

Binding of ligands, including RGD-containing peptides, to the platelet fibrinogen receptor, integrin alphaIIb beta3 has been reported to cause outside-in signals, which result in clustering of occupied receptors and changes in conformation of the receptor and its cytoplasmic tails. Thus, the peptides that are usually used as inhibitors may function as partial agonists. Binding of ligand, fibrinogen or polymerizing fibrin, to platelets with activated alphaIIbbeta3 causes decreases in phosphatidylinositol 4,5-bisphosphate (PIP ), which may affect actin organization. Whether or not binding to unactivated platelets of the peptide RGDS, the fibrinogen gamma -chain C-terminal dodecapeptide (H12), or a high affinity RGD mimetic SC-54701B affects phosphoinositide metabolism was tested. Although incubation of RGDS (230 microM), dodecapeptide (400 microM) or SC-54701B (10 microM) with platelets for 2 min caused trends towards decreases in PIP , no significant decreases were found. As a positive control, 2 SC-54701B was shown to inhibit the decrease in PIP in ADP-stimulated platelets at concentrations consistent 2 with the reported IC50 of 0.12 microM. Thus, binding of peptides or the high affinity RGD mimetic does not 50 generate a sufficient signal to affect the signalling pathway that is involved phosphoinositide metabolism.

Journal Article↗
PubMed2003

Pharmacological inhibition of plasma coagulation and platelet activation during experimental long-term perfusion.

OBJECTIVE: During extracorporeal circulation, initial contact between blood and the artificial surface of the circuit induces an overall activation of the hemostatic system. The objective of this study was to investigate the combined effect of epoprostenol (PGI (2) ), nitric oxide (NO) and nafamostat mesilate (FUT-175, a serine protease inhibitor), on plasma coagulation and platelet activation during experimental long-term perfusion. DESIGN: Two identical extracorporeal life support (ECLS) circuits were primed with fresh, heparinized human blood, and circulated for 24 h. FUT was given with a bolus dose of 85 mg/l blood at the initiation of the experiment and thereafter as a continuous infusion of 14 mg/l/h. PGI (2), at a rate of 2.4 microg/l/h, was also administered to the experimental circuit, and 120 ppm NO gas was added to the oxygenator sweep gas. The other circuit was used as a control. RESULTS: Higher platelet count and platelet membrane expression of GPIb were found in the experimental circuits as compared with control circuits. The levels of thrombin/antithrombin III complex (TAT) and prothrombin fragment 1 + 2 (F1 + 2) increased significantly over time in the control circuits but remained low in the experimental circuits. Plasma levels of plasminogen activator inhibitor 1 (PAI-1) decreased rapidly in both circuits but were higher in the control circuits at each time point studied. CONCLUSION: The activation of platelets and of the coagulation system encountered during extracorporeal perfusion is consistently inhibited by a combination of PGI (2), NO and FUT-175. The combination of these drugs appears to be more effective than each drug separately.

Antithrombin III↗
PubMed2002

Enhanced expansion and maturation of megakaryocytic progenitors by fibronectin.

BACKGROUND: Megakaryopoiesis occurs as a result of a complex interaction between hemopoietic cells, stromal cells, hemopoietic growth factors and extracellular matrix (ECM). Megakaryocyte growth and development factor (MGDF) or thrombopoietin is the main growth factor that induces megakaryocyte commitment. The role of adhesion proteins in the generation of megakaryocytic progenitors has not been well studied. METHODS: We isolated CD34(+) cells from umbilical cord (UC) blood and cultured them in serum-free medium containing IL-3, IL-6, SCF and MGDF, with or without fibronectin. Cultures were sampled at Days 4 and 8 for cell count, immunophenotyping by flow cytometry, and megakaryocyte colony-forming units (CFU-Mk) assay. RESULTS: Fibronectin in synergy with MGDF increased both total and CD34(+) cell count. Immunophenotyping of the CD34(+) and CD34(-) cells showed that the percentage expression of CD61 and CD41a on CD34(-) cells was increased by fibronectin compared with CD34(+) cells by Day 8 of culture. The CFU-Mk assay confirmed that fibronectin increased generation of megakaryocytic progenitors by 2.4-fold by Day 8 of culture, but the absolute number was less than that of Day 4 culture. The cells in the CFU-Mk colonies derived from culture containing fibronectin were larger than those from cytokine-only culture. CONCLUSION: It was concluded that the addition of fibronectin to the culture system enhanced MGDF, induced ex vivo expansion of megakaryocytic progenitors from CD34(+) cells, as well as increasing their maturation towards later stages of megakaryocyte differentiation.

Antibodies, Monoclonal↗
PubMed2003

Expansion of HPCs from cord blood in a novel 3D matrix.

BACKGROUND: An optimal system for the expansion of pluripotent HPCs would ideally eliminate the use of cytokines and animal-derived serum. We have shown previously that a 3D, tantalum-coated porous biomaterial (Cytomatrix) supports the maintenance and expansion of human BM HPCs in the absence of cytokines. METHODS: Umbilical cord blood (UCB) derived HPC were cultured in the Cytomatrix in the absence of exogenous cytokines. Phenotype was determined using FACS. Colony-forming units (CFU) activity was evaluated. Engraftment capacity was evaluated by transplanting the expanded cells into non-obese diabetic (NOD)/SCID mice. RESULTS: We describe the expansion of HPCs from UCB using the Cytomatrix system. When UCB-derived CD34(+) cells were cultured in the Cytomatrix system for 2 weeks we observed an increase in the number of nucleated cells (3-fold) and CFU (2.6-fold). The number of CD45(+) and CD34(+) cells both increased three-fold. Trends demonstrated an increase in the frequency of CD34(+)C38(-) cells, and an increase in both CD34(+)C33(+) cells and CD34(+)C61(+) cells. No expansion of T or B lymphocytes was observed. When expanded UCB cells from the Cytomatrix were injected into sub-lethally irradiated NOD/SCID mice, human cells were detected in the murine peripheral blood and BM 6 weeks post-transplantation. DISCUSSION: This unique approach to the expansion of UCB cells in a serum-free, cytokine-free environment may provide expansion of HPCs with multi-lineage engraftment capability that could be used clinically.

ADP-ribosyl Cyclase↗
PubMedDate not supplied

Decreased expression of alpha3 and beta1 integrin subunits is responsible for differentiation-associated changes in cells behavior in terminally differentiated human oral keratinocytes.

Primary normal human oral keratinocytes (NHOKs) terminally differentiate in serial subculture. To investigate whether this subculture-induced differentiation of NHOKs affects integrin expression and cell-matrix interaction, we studied the expression levels of integrin subunits and cellular response to the extracellular matrix (ECM) proteins in NHOKs at different population doublings. The phosphorylation statuses of focal adhesion kinase (FAK), extracellular signal regulated kinase (ERK), p38, and c-Jun amino-terminal kinase (JNK) were also determined in NHOK cells cultured on ECM proteins, to evaluate the functions of integrins with respect to cellular responses to ECM proteins. The expression levels of alpha3 and beta1 integrin subunits progressively decreased in NHOKs undergoing terminal differentiation. The ability of NHOKs to spread upon laminin and type I collagen significantly decreased in terminally differentiated oral keratinocytes. Keratinocyte migration was significantly increased on type I collagen for terminally differentiated NHOKs. Similar results were seen following preincubation of rapidly proliferating NHOKs with function-blocking antibodies to alpha3 or beta1 integrin subunit. In contrast, fibronectin had no effect on cellular responses in NHOKs, which were almost negligible in the expression levels of alpha5 integrin subunits. The extent of FAK phosphorylation in terminally differentiated NHOKs was notably lower than that of rapidly proliferating cells, but was enhanced in terminally differentiated cells that were cultured on type I collagen. Our results indicate that decreased expression of alpha3 and beta1 integrin subunits is responsible for differentiation-associated changes in cells behavior in terminally differentiated oral keratinocytes. Our data also show that the abrogation of the alpha5beta1 integrin function caused by omitting alpha5 subunit is linked to the loss of a cell-fibronectin interaction in human oral keratinocytes.

Antibodies, Monoclonal↗
PubMed1996

Integrin-associated protein immunoglobulin domain is necessary for efficient vitronectin bead binding.

Integrin-associated protein (IAP/CD47) is physically associated with the alpha v beta 3 vitronectin (Vn) receptor and a functionally and immunologically related integrin on neutrophils (PMN) and monocytes. Anti-IAP antibodies inhibit multiple phagocyte functions, including Arg-Gly-Asp (RGD)-initiated activation of phagocytosis, chemotaxis, and respiratory burst; PMN adhesion to entactin; and PMN transendothelial and transepithelial migration at a step subsequent to tight intercellular adhesion. Anti-IAP antibodies also inhibit binding of Vn-coated particles to many cells expressing alpha v beta 3. However, prior studies with anti-IAP did not directly address IAP function because they could not distinguish between IAP blockade and antibody-induced signaling effects on cells. To better determine the function of IAP, we have characterized and used an IAP-deficient human cell line. Despite expressing alpha v integrins, these cells do not bind Vn-coated particles unless transfected with IAP expression constructs. Increasing the level of alpha v beta 3 expression or increasing Vn density on the particle does not overcome the requirement for IAP. All known splice variants of IAP restore Vn particle binding equivalently. Indeed, the membrane-anchored IAP Ig variable domain suffices to mediate Vn particle binding in this system, while the multiply membrane-spanning and cytoplasmic domains are dispensable. In all cases, adhesion to a Vn-coated surface and fibronectin particle binding through alpha 5 beta 1 fibronectin receptors are independent of IAP expression. These data demonstrate that some alpha v integrin ligand-binding functions are IAP independent, whereas others require IAP, presumably through direct physical interaction between its Ig domain and the integrin.

Amino Acid Sequence↗
PubMed1998

Growth factor-dependent activation of alphavbeta3 integrin in normal epithelial cells: implications for tumor invasion.

Integrin activation is a multifaceted phenomenon leading to increased affinity and avidity for matrix ligands. To investigate whether cytokines produced during stromal infiltration of carcinoma cells activate nonfunctional epithelial integrins, a cellular system of human thyroid clones derived from normal glands (HTU-5) and papillary carcinomas (HTU-34) was employed. In HTU-5 cells, alphavbeta3 integrin was diffused all over the membrane, disconnected from the cytoskeleton, and unable to mediate adhesion. Conversely, in HTU-34 cells, alphavbeta3 was clustered at focal contacts (FCs) and mediated firm attachment and spreading. alphavbeta3 recruitment at FCs and ligand-binding activity, essentially identical to those of HTU-34, occurred in HTU-5 cells upon treatment with hepatocyte growth factor/scatter factor (HGF/SF). The HTU-34 clone secreted HGF/SF and its receptor was constitutively tyrosine phosphorylated suggesting an autocrine loop responsible for alphavbeta3 activated state. Antibody-mediated inhibition of HGF/SF function in HTU-34 cells disrupted alphavbeta3 enrichment at FCs and impaired adhesion. Accordingly, activation of alphavbeta3 in normal cells was produced by HTU-34 conditioned medium on the basis of its content of HGF/SF. These results provide the first example of a growth factor-driven integrin activation mechanism in normal epithelial cells and uncover the importance of cytokine-based autocrine loops for the physiological control of integrin activation.

Antigens, CD↗
PubMed2000

Restructuring of focal adhesion plaques by PI 3-kinase. Regulation by PtdIns (3,4,5)-p(3) binding to alpha-actinin.

Focal adhesions are an elaborate network of interconnecting proteins linking actin stress fibers to the extracellular matrix substrate. Modulation of the focal adhesion plaque provides a mechanism for the regulation of cellular adhesive strength. Using interference reflection microscopy, we found that activation of phosphoinositide 3-kinase (PI 3-kinase) by PDGF induces the dissipation of focal adhesions. Loss of this close apposition between the cell membrane and the extracellular matrix coincided with a redistribution of alpha-actinin and vinculin from the focal adhesion complex to the Triton X-100-soluble fraction. In contrast, talin and paxillin remained localized to focal adhesions, suggesting that activation of PI 3-kinase induced a restructuring of the plaque rather than complete dispersion. Furthermore, phosphatidylinositol (3,4, 5)-trisphosphate (PtdIns (3,4,5)-P(3)), a lipid product of PI 3-kinase, was sufficient to induce restructuring of the focal adhesion plaque. We also found that PtdIns (3,4,5)-P(3) binds to alpha-actinin in PDGF-treated cells. Further evidence demonstrated that activation of PI 3-kinase by PDGF induced a decrease in the association of alpha-actinin with the integrin beta subunit, and that PtdIns (3,4,5)-P(3) could disrupt this interaction in vitro. Modification of focal adhesion structure by PI 3-kinase and its lipid product, PtdIns (3,4,5)-P(3), has important implications for the regulation of cellular adhesive strength and motility.

Actinin↗
PubMed2001

Apoptosis of adherent cells by recruitment of caspase-8 to unligated integrins.

Integrin-mediated adhesion promotes cell survival in vitro, whereas integrin antagonists induce apoptosis of adherent cells in vivo. Here, we demonstrate that cells adherent within a three-dimensional extracellular matrix undergo apoptosis due to expression of unligated integrins, the beta subunit cytoplasmic domain, or its membrane proximal sequence KLLITIHDRKEF. Integrin-mediated death requires initiator, but not stress, caspase activity and is distinct from anoikis, which is caused by the loss of adhesion per se. Surprisingly, unligated integrin or beta integrin tails recruit caspase-8 to the membrane, where it becomes activated in a death receptor-independent manner. Integrin ligation disrupts this integrin-caspase containing complex and increases survival, revealing an unexpected role for integrins in the regulation of apoptosis and tissue remodeling.

Animals↗
PubMed2001

Expansion of megakaryocyte precursors and stem cells from umbilical cord blood CD34+ cells in collagen and liquid culture media.

Umbilical cord blood (UCB) is now commonly used as a source of stem cells for hematopoietic reconstitution following myeloablative therapy in patients with a variety of diseases. Although UCB is a rich source of stem cells, platelet engraftment occurs at a median of 71 days which is significantly prolonged compared to allogeneic bone marrow. The number of megakaryocyte (MK) precursors in stem cell harvests appears to correlate inversely with the time to platelet engraftment. In an effort to increase the number of platelet precursors, we cultured CD34-selected cord blood mononuclear cells (MNC) in serum-free collagen medium with numerous cytokine combinations. The cells were cultured with four cytokines: interleukin-3 (IL-3), thrombopoietin (TPO), stem cell factor (SCF), and Flt-3); five cytokines, IL-3, TPO, SCF, Flt-3 plus granulocyte-macrophage colony-stimulating factor (GM-CSF), or erythropoietin (Epo); or all six cytokines in combination. After 16 days, significant expansion of MK precursors (CD41(+)) and stem cells (CD34(+) and AC133(+) cells) were seen in cells cultured in IL-3, TPO, SCF, and Flt-3 with or without GM-CSF compared to the combinations that contained Epo (p < 0.05). Similar studies were performed using liquid culture medium, and after 14 days the number of MNCs, CD34(+), AC133(+), CD41(+), and CD61(+) cells were higher in the UCB cells cultured in IL-3, TPO, SCF, and Flt-3 compared to those cultured with those four cytokines plus GM-CSF. These results demonstrate that UCB stem cells can be effectively expanded ex vivo and enriched with platelet precursors using TPO, SCF, Flt-3, and IL-3, whereas the addition of Epo and GM-CSF is unnecessary.

AC133 Antigen↗
PubMed2005

Platelet-derived growth factor enhances expansion of umbilical cord blood CD34+ cells in contact with hematopoietic stroma.

Stem cell expansion remains an elusive but highly desirable goal. Here we show that platelet-derived growth factor (PDGF), along with cultured endothelial or stromal cells, significantly enhances expansion of human CD34+ cells in vitro. In media supplemented with thrombopoietin, stem cell factor, flt-3 ligand, and granulocyte-colony stimulating factor, CD34+ cells, as well as CFU-GM, BFU-CFU-E, CFU-GEMM, and CFU-MK, increased by 34.3-, 138-, 59.7-, 38.4-, and 86.0-fold, respectively. Co-culturing of CD34+ cells with cultured stromal cells or human umbilical cord vein endothelial cells (HUVECs) greatly enhanced expansion efficiency. The presence of PDGF (50 ng/ml) further augmented expansion, such that increases of 77.0-, 262-, 90.0-, 93.0-, and 200-fold, respectively, were achieved. Six weeks after infusion of expanded cells into NOD/SCID mice, human CD45+ cells were detected in recipients' bone marrow, spleen, and peripheral blood. Our results provide a rationale for development of a stem cell expansion protocol for clinical applications.

Animals↗
PubMed1998

beta1-integrin cytoplasmic subdomains involved in dominant negative function.

The beta1-integrin cytoplasmic domain consists of a membrane proximal subdomain common to the four known isoforms ("common" region) and a distal subdomain specific for each isoform ("variable" region). To investigate in detail the role of these subdomains in integrin-dependent cellular functions, we used beta1A and beta1B isoforms as well as four mutants lacking the entire cytoplasmic domain (beta1TR), the variable region (beta1COM), or the common region (beta1 deltaCOM-B and beta1 deltaCOM-A). By expressing these constructs in Chinese hamster ovary and beta1 integrin-deficient GD25 cells (Wennerberg et al., J Cell Biol 132, 227-238, 1996), we show that beta1B, beta1COM, beta1 deltaCOM-B, and beta1 deltaCOM-A molecules are unable to support efficient cell adhesion to matrix proteins. On exposure to Mn++ ions, however, beta1B, but none of the mutants, can mediate cell adhesion, indicating specific functional properties of this isoform. Analysis of adhesive functions of transfected cells shows that beta1B interferes in a dominant negative manner with beta1A and beta3/beta5 integrins in cell spreading, focal adhesion formation, focal adhesion kinase tyrosine phosphorylation, and fibronectin matrix assembly. None of the beta1 mutants tested shows this property, indicating that the dominant negative effect depends on the specific combination of common and B subdomains, rather than from the absence of the A subdomain in the beta1B isoform.

Actinin↗
PubMed2006

Effects of antiplatelet components of tomato extract on platelet function in vitro and ex vivo: a time-course cannulation study in healthy humans.

BACKGROUND: Natural antithrombotic agents that influence platelet function are of potential interest for primary prevention of cardiovascular disease. Previous reports showed that tomato extracts inhibit platelet aggregation in vitro, but little is known of the active components, their mode of action, or their efficacy in vivo. OBJECTIVE: The objectives of the study were to examine the antiplatelet activity of specific tomato components by in vitro experimentation and to establish their ex vivo efficacy in healthy humans. DESIGN: The mechanisms of action of antiplatelet components isolated from tomato extracts were examined in vitro. A 7-h time-course study was carried out in cannulated human subjects (n = 23) to determine the ex vivo efficacy of a supplement drink containing tomato extract and the onset and duration of antiplatelet effects. RESULTS: The inhibition of ADP-, collagen-, thrombin-, and arachidonate-mediated platelet aggregation by tomato extract components appears to be linked to the inhibition of glycoprotein IIb/IIIa and platelet secretory mechanisms. We found a significant inhibition of baseline platelet function, from 2.9 +/- 1.4% (optimal ADP concentrations; P = 0.03) to 20.0 +/- 4.9% (suboptimal ADP concentrations; P < 0.001), 3 h after supplementation with a dose of tomato extract equivalent to 6 tomatoes. The observed effects persisted for >12 h. Coagulation variables were not affected. CONCLUSIONS: The ingestion of tomato components with in vitro antiplatelet activity significantly affects ex vivo platelet function. The reported cardioprotective effects of tomatoes are potentially linked to a modulation of platelet function.

Adult↗
PubMed1995

Fetal bleeding in neonatal alloimmune thrombocytopenia mediated by anti-PlAl is not associated with inhibition of fibrinogen binding to platelet GPIIb/IIIa.

Antibody directed against the platelet-specific alloantigen, PlAl, is the most frequently reported cause of two syndromes, post-transfusion purpura (PTP), and neonatal alloimmune thrombocytopenia (NAIT). Numerous reports have indicated that anti-PlAl also has the ability to block certain responses of platelets to stimulation, including fibrinogen binding, platelet aggregation, and serotonin release. Because the PlAl epitope is located on platelet membrane glycoprotein (GP) IIb/IIIa that also contains the fibrinogen receptor, these effects may be mediated by antibody binding at or near the fibrinogen receptor site. This study examines the capacity of anti-PlAl from patients with PTP and from mothers of infants affected by the NAIT to block the binding of radio-labeled fibrinogen to washed human platelets stimulated by ADP and epinephrine. In six of the seven PTP patients, there was inhibition of fibrinogen binding, ranging from 28% to 84% inhibition. In contrast, all anti-PlAl sera from nine mothers of infants with NAIT, including four with intracranial hemorrhage, failed to inhibit fibrinogen binding. Despite the generally higher anti-PlAl titers of the PTP sera, the ability to inhibit fibrinogen binding did not appear attributable to antibody titers. These results suggest that interference with fibrinogen binding to platelets by maternal anti-PlAl does not underlie the increased risk of bleeding in NAIT, whereas inhibitory activity directed against fibrinogen binding appears to be a characteristic feature of the sera from PTP patients.

Antigens, Human Platelet↗