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• W1990595551 abstract "Upon activation, platelets store and release large amounts of the peptide transforming growth factor β1 (TGFβ1). The released TGFβ1 can then act on nearby vascular cells to mediate subsequent vessel repair. In addition, TGFβ1 may circulate to bone marrow and regulate megakaryocyte activity. It is not known what effect, if any, TGFβ1 has on platelets. Adult TGFβ1-deficient mice exhibit thrombocythemia and a mild bleeding disorder that is shown to result from faulty platelet aggregation. TGFβ1-deficient platelets are shown to contain functional receptors, and preincubation with recombinant TGFβ1 improves aggregation, demonstrating that TGFβ1 plays an active role in platelet aggregation. TGFβ1-deficient platelets fail to retain bound fibrinogen in response to aggregation agonists, but they possess normal levels of the αIIb/β3 fibrinogen receptor. Signaling from agonist receptors is normal because the platelets change shape, produce thromboxane A2, and present P-selectin in response to stimulation. Consequently, activation and maintenance of αIIb/β3 into a fibrinogen-binding conformation is impaired in the absence of TGFβ1. 4-Phorbol 12-myristate 13-acetate treatment and protein kinase C activity measurements suggest a defect downstream of protein kinase C in its activation cascade. Because platelets lack nuclei, these data demonstrate for the first time a non-transcriptionally mediated TGFβ1 signaling pathway that enhances the activation and maintenance of integrin function. Upon activation, platelets store and release large amounts of the peptide transforming growth factor β1 (TGFβ1). The released TGFβ1 can then act on nearby vascular cells to mediate subsequent vessel repair. In addition, TGFβ1 may circulate to bone marrow and regulate megakaryocyte activity. It is not known what effect, if any, TGFβ1 has on platelets. Adult TGFβ1-deficient mice exhibit thrombocythemia and a mild bleeding disorder that is shown to result from faulty platelet aggregation. TGFβ1-deficient platelets are shown to contain functional receptors, and preincubation with recombinant TGFβ1 improves aggregation, demonstrating that TGFβ1 plays an active role in platelet aggregation. TGFβ1-deficient platelets fail to retain bound fibrinogen in response to aggregation agonists, but they possess normal levels of the αIIb/β3 fibrinogen receptor. Signaling from agonist receptors is normal because the platelets change shape, produce thromboxane A2, and present P-selectin in response to stimulation. Consequently, activation and maintenance of αIIb/β3 into a fibrinogen-binding conformation is impaired in the absence of TGFβ1. 4-Phorbol 12-myristate 13-acetate treatment and protein kinase C activity measurements suggest a defect downstream of protein kinase C in its activation cascade. Because platelets lack nuclei, these data demonstrate for the first time a non-transcriptionally mediated TGFβ1 signaling pathway that enhances the activation and maintenance of integrin function. transforming growth factor β1 protein transforming growth factor β1 gene severe combined immunodeficiency thromboxane A2 (1S-[1α,2β(5Z),3α(1E,3R*),4α])-7-(3-[3-hydroxy-4-(4′-iodophenoxy)-1-butenyl]-7-oxabicyclo[2.2.1]heptan-2-yl)-5-heptenoic acid platelet-rich plasma platelet-poor plasma 4-phorbol 12-myristate 13-acetate protein kinase C phosphoinositol 3-kinase fluorescein isothiocyanate human iliac vein endothelial cells Platelets function in hemostasis following vascular injury by arresting blood loss at the site of injury and delivering a variety of molecules to the injured vessel wall. Normally inactive, platelets adhere to vascular matrices exposed during injury and become activated (1Vlodavsky I. Eldor A. HyAm E. Atzom R. Fuks Z. Thromb. Res. 1982; 28: 179-191Abstract Full Text PDF PubMed Scopus (52) Google Scholar, 2Turitto V.T. Weiss H.J. Zimmerman T.S. Sussman I.I. Blood. 1985; 65: 823-831Crossref PubMed Google Scholar). Storage granules are then mobilized to the platelet surface, releasing a variety of molecules that mediate subsequent hemostatic events (3Rink T.J. Smith S.W. Tsien R.Y. FEBS Lett. 1982; 148: 21-26Crossref PubMed Scopus (387) Google Scholar). TGFβ11 is stored in large amounts as a latent peptide in the secretory α-granules of circulating platelets and is one of the molecules released during platelet activation (4Assoian R.K. Komoriya A. Meyers C.A. Miller D.M. Sporn M.B. J. Biol. Chem. 1983; 258: 7155-7160Abstract Full Text PDF PubMed Google Scholar). Active platelets can release enough TGFβ1 to raise the local concentration of TGFβ1 to as much as 40 ng/ml at the injury site and in the developing thrombus (5Grainger D.J. Wakefield L. Bethell H.W. Farndale R.W. Metcalfe J.C. Nat. Med. 1995; 1: 932-937Crossref PubMed Scopus (194) Google Scholar). TGFβ1 can influence vessel repair through regulation of endothelial cell function (6Gamble J.R. Vadas M.A. Science. 1988; 242: 97-99Crossref PubMed Scopus (184) Google Scholar, 7Heimark R.L. Twardzik D.R. Schwartz S.M. Science. 1986; 233: 1078-1080Crossref PubMed Scopus (336) Google Scholar), smooth muscle cell differentiation (8Bjorkerud S. Arterioscler. Thromb. 1991; 11: 892-902Crossref PubMed Google Scholar, 9Shi Y. O'Brien J.E.J. Fard A. Zalewski A. Arterioscler. Thromb. Vasc. Biol. 1996; 16: 1298-1305Crossref PubMed Scopus (124) Google Scholar), and vessel wall remodeling (10Nabel E.G. Shum L. Pompili V.J. Yang Z.Y. San H. Shu H.B. Liptay S. Gold L. Gordon D. Derynck R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10759-10763Crossref PubMed Scopus (343) Google Scholar, 11Lawrence R. Hartmann D.J. Sonenshein G.E. J. Biol. Chem. 1994; 269: 9603-9609Abstract Full Text PDF PubMed Google Scholar). It is not known whether TGFβ1 can also regulate platelet activity. In nucleated cells, members of the Smad gene family are believed the primary mediators of intracellular signaling from the TGFβ receptor types I and II (12Heldin C.H. Miyazono K. ten Dijke P. Nature. 1997; 390: 465-471Crossref PubMed Scopus (3358) Google Scholar, 13Kretzschmar M. Massague J. Curr. Opin. Genet. Dev. 1998; 8: 103-111Crossref PubMed Scopus (433) Google Scholar). SMADs are able to bind DNA (14Zawel L. Dai J.L. Buckhaults P. Zhou S. Kinzler K.W. Vogelstein B. Kern S.E. Mol. Cell. 1998; 1: 611-617Abstract Full Text Full Text PDF PubMed Scopus (892) Google Scholar, 15Shi Y. Wang Y.F. Jayaraman L. Yang H. Massague J. Pavletich N.P. Cell. 1998; 94: 585-594Abstract Full Text Full Text PDF PubMed Scopus (612) Google Scholar) and can regulate transcription of TGFβ1-responsive genes (16Dennler S. Itoh S. Vivien D. ten Dijke P. Huet S. Gauthier J.M. EMBO J. 1998; 17: 3091-3100Crossref PubMed Scopus (1588) Google Scholar, 17Vindevoghel L. Kon A. Lechleider R.J. Uitto J. Roberts A.B. Mauviel A. J. Biol. Chem. 1998; 273: 13053-13057Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). However, the type I receptor can interact with other molecules such as the α subunit of farnesyltransferase (18Kawabata M. Imamura T. Miyazono K. Engel M.E. Moses H.L. J. Biol. Chem. 1995; 270: 29628-29631Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Additionally, TGFβ1 can stabilize protein levels in epithelial cells (19Sandhu C. Garbe J. Bhattacharya N. Daksis J. Pan C.H. Yaswen P. Koh J. Slingerland J.M. Stampfer M.R. Mol. Cell. Biol. 1997; 17: 2458-2467Crossref PubMed Google Scholar). These findings suggest that there are possible transcription-independent signaling pathways for TGFβ1 (19Sandhu C. Garbe J. Bhattacharya N. Daksis J. Pan C.H. Yaswen P. Koh J. Slingerland J.M. Stampfer M.R. Mol. Cell. Biol. 1997; 17: 2458-2467Crossref PubMed Google Scholar). The primary defect in the TGFβ1-deficient mouse is a severe multifocal inflammatory disease resulting in death by the 3rd week after birth (20Shull M.M. Ormsby I. Kier A.B. Pawlowski S. Diebold R.J. Yin M. Allen R. Sidman C. Proetzel G. Calvin D. Annunziata N. Doetschman T. Nature. 1992; 359: 693-699Crossref PubMed Scopus (2653) Google Scholar, 21Kulkarni A.B. Huh C.G. Becker D. Geiser A. Lyght M. Flanders K.C. Roberts A.B. Sporn M.B. Ward J.M. Karlsson S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 770-774Crossref PubMed Scopus (1663) Google Scholar, 22Boivin G.P. O'Toole B.A. Ormsby I.E. Diebold R.J. Eis M.J. Doetschman T.C. Kier A.B. Am. J. Pathol. 1995; 146: 276-288PubMed Google Scholar). Genetic combination of Tgfb1 knockout and Scid alleles eliminates the inflammation and increases longevity by 3–5 months (23Diebold R.J. Eis M.J. Yin M. Ormsby I. Boivin G.P. Darrow B.J. Saffitz J.E. Doetschman T. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 12215-12219Crossref PubMed Scopus (233) Google Scholar), thereby permitting investigation of adult phenotypes primary to the absence of TGFβ1 and not compromised by secondary effects resulting from inflammation. Scid Tgfb1 −/− mice have thrombocythemia and a mild bleeding disorder that is associated with faulty platelet aggregation resulting from a failure to sustain fibrinogen binding. Preincubation with active TGFβ1 improves platelet aggregation, indicating the presence of a TGFβ1 signaling pathway in platelets. Because TGFβ1 signaling in platelets is necessarily independent of transcription, these results demonstrate the existence of a non-transcriptional TGFβ1 signaling pathway that mediates platelet function by affecting the state of integrin activation. Tgfb1 −/− mice are on a mixed strain background of 129S2/SvPas × BL/Swiss × CF-1 (approximately 25%, 25%, and 50%, respectively) (20Shull M.M. Ormsby I. Kier A.B. Pawlowski S. Diebold R.J. Yin M. Allen R. Sidman C. Proetzel G. Calvin D. Annunziata N. Doetschman T. Nature. 1992; 359: 693-699Crossref PubMed Scopus (2653) Google Scholar). To place the null Tgfb1 allele on an immune-compromised background, they were back-crossed for three generations to C3H mice homozygous for theScid allele (23Diebold R.J. Eis M.J. Yin M. Ormsby I. Boivin G.P. Darrow B.J. Saffitz J.E. Doetschman T. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 12215-12219Crossref PubMed Scopus (233) Google Scholar). For all strains, experimental animals were obtained through Scid Tgfb1 +/− ×Scid Tgfb1 +/− matings. When possible, Scid Tgfb1 +/+ andScid Tgfb1 +/− siblings of Scid Tgfb1 −/− offspring were used as controls. Blood samples were obtained by cardiac puncture of anesthetized mice using EDTA (10 mm) as an anticoagulant. Platelets in diluted whole blood were counted in duplicate with a thin hemocytometer under 100× phase microscopy. Bone marrow was collected from bare femurs and tibiae of mice by flushing with a 10-ml empty syringe fitted with a 21-gauge needle rinsed with 10 mm EDTA. Drops of marrow were smeared on glass slides and stained with Wright and Giemsa stains. The average number of megakaryocytes per 10× field covering the entire smear was determined. Bleeding times were determined by a previously reported method (24Oyekan A.O. Onabanjo A.O. Haemostasis. 1991; 21: 360-369PubMed Google Scholar). Briefly, the base of each tail was treated with a depilatory, cleaned with alcohol, and air-dried. A puncture was made along the lateral side of the tail using a sterile lancet with a blade depth of 2.5 mm and width of 1.5 mm (Microlance; Becton and Dickinson). The average bleeding time was measured by constant observation under a dissecting microscope while blotting with filter paper every 15 s so that the bleeding stop could be accurately observed. Measurements from three separate punctures were made for each mouse. Blood samples were collected with a butterfly needle (25 g × 3/8, 3.5-inch tubing, Abbott Laboratories) and a 3-ml syringe containing 3.8% sodium citrate through a clean abdominal vena cava puncture. Typically, we obtained 0.6–0.8 ml of blood from Scid Tgfb1 +/+ mice and 0.5–0.6 of blood fromScid Tgfb1 −/− mice. A blood:citrate ratio of approximately 6:1 was used in the collections by using 100 μl and 80 μl of citrate for Scid Tgfb1 +/+ and Scid Tgfb1 −/− mice, respectively. Platelet-rich plasma (PRP) was prepared by centrifugation of blood at 150 ×g for 15 min. The remaining portion was then centrifuged 10 min at 2000 rpm to recover platelet-poor plasma (PPP). Previous experience indicated that samples showing signs of red blood cell hemolysis did not perform well and thus were discarded. PRP recovered from 3–5 mice were pooled and the platelet concentration adjusted to 3 × 108 platelets/ml with PPP. Aggregation experiments were performed with 225 μl of PRP at 37 °C with constant stirring in an optical aggregometer (Chrono-log Corp.). Aggregation was initiated by adding 25 μl of 10× agonist. Experiments involving thrombin included 0.25 mmglycyl-prolyl-arginyl-proline peptide (GPRP, Sigma), to prevent fibrin clot formation (25Warkentin T.E. Powling M.J. Hardisty R.M. Br. J. Haematol. 1990; 76: 387-394Crossref PubMed Scopus (62) Google Scholar). For experiments involving preincubation with TGFβ1, 250 μl of PRP was incubated at room temperature for at least 1 h with active recombinant human TGFβ1 (R&D Systems) prior to performing the aggregation experiment. Detection of bound fibrinogen was carried out essentially as described previously (25Warkentin T.E. Powling M.J. Hardisty R.M. Br. J. Haematol. 1990; 76: 387-394Crossref PubMed Scopus (62) Google Scholar). 2 × 106 platelets in 3–5 μl of PRP were added to a tube containing 50 μl of HEPES buffer (10 mm HEPES, 145 mm NaCl, 5 mm KCl, 1 mmMgSO4, pH7.4) and 5 μl of FITC-conjugated polyclonal rabbit anti-human fibrinogen antibody (Dako Patts) with or without 5 μl of ADP (10 μm final), the radiolabeled TXA2 mimetic ((1S-[1α,2β(5Z),3α(1E,3R*),4α])- 7-(3-[3-hydroxy-4-(4′-iodophenoxy)-1-butenyl]-7-oxabicyclo[2.2.1]heptan-2-yl)-5-heptenoic acid (I-BOP), 10 μm final) or thrombin (0.4 units/ml final). After incubation in the dark at room temperature for 30 min, 0.5 ml of 0.2% formalin was added. To follow the release of bound fibrinogen by platelets, the anti-fibrinogen antibody was added at 0, 5, 10, and 15 min after stimulation by ADP to separate aliquots of PRP diluted in HEPES buffer. The experiment continued as described above. For detection of P-selectin, 1 μg of a FITC-conjugated monoclonal rat antibody to mouse P-selectin (PharMingen) was used in place of the anti-fibrinogen antibody and was present at the time of agonist addition. Samples stimulated with thrombin contained 0.25 mm GPRP. Analysis was performed on a FACSTAR instrument (Becton Dickinson) using the FACSTAR Lysis II software. Specificity of the anti-fibrinogen antibody for fibrinogen was determined in two ways. Wild type platelets were activated in the presence of a FITC-conjugated rat anti-mouse T lymphocyte antibody (Thy1.2, PharMingen) to control for nonspecific binding of IgG by platelets. In the other control experiments, an excess of non-immune rabbit IgG was included with the anti-fibrinogen antibody during platelet activation. Activated wild type mouse platelets gave a slightly higher background signal with the anti-Thy1 antibody than resting platelets incubated with the anti-fibrinogen antibody. The excess IgG did not affect the positive fluorescent peak typically obtained with activated wild type platelets (data not shown). PAC-1, an antibody that specifically recognizes the activated form of human αIIb/β3 (26Shattil S.J. Hoxie J.A. Cunningham M. Brass L.F. J. Biol. Chem. 1985; 260: 11107-11114Abstract Full Text PDF PubMed Google Scholar) did not interact with the mouse platelets (from eitherTgfb1 +/+ or Tgfb1 −/−mice) used in our experiments. PRP containing 2 × 107 platelets was incubated in the presence or absence of 10 μm ADP at 37 °C for 10 min without stirring. Samples were immediately centrifuged and the supernatants transferred to fresh tubes. An aliquot (25 μl) from each supernatant and plasma collected during preparation of the PRP were assayed for thromboxane B2 levels using a colorimetric enzyme immunoassay (Biotrak, Amersham Pharmacia Biotech) according to manufacturer's instructions. Identification of platelet surface TGFβ1-binding proteins was performed by affinity cross-linking of125I-TGFβ1 with disuccinimidyl suberate (27Massague J. Like B. J. Biol. Chem. 1985; 260: 2636-2645Abstract Full Text PDF PubMed Google Scholar). Platelets were collected by venipuncture and washed twice with cold binding buffer (128 mm NaCl, 5 mm KCl, 1.2 mm CaCl2, 5 mm MgSO4, 50 mm HEPES, and 5 mg/ml bovine serum albumin at pH 7.5) containing 1 μm prostacyclin to prevent activation. Washed platelets (3 × 107) were incubated at 4 °C for 2.5 h with 150 pm125I-TGFβ1 in 0.5 ml of binding buffer. All incubations were performed in siliconized microcentrifuge tubes to minimize binding of the125I-TGFβ1 to tube surfaces. Following three washes in cold binding buffer, platelets were incubated in 0.5 ml of binding buffer with 0.25 mm disuccinimidyl suberate at 4 °C for 15 min. The cross-linking reaction was stopped by washing the platelets twice in cold stop buffer (0.25 m sucrose, 1 mmEDTA, 10 mm Tris, pH 7.4) containing protease inhibitors. Each platelet pellet was solubilized for 40 min at 4 °C in 20 μl of detergent buffer (1% Triton X-100, 1 mm EDTA, 125 mm NaCl, 10 mm Tris, pH 7.4) containing protease inhibitors. Samples were centrifuged to remove the insoluble material and the supernatants boiled for 5 min in an equal volume of SDS loading buffer under reducing (50 mm dithiothreitol) or non-reducing (no dithiothreitol) conditions. Samples were separated on a 7% polyacrylamide gel electrophoresis gel, dried, and exposed to x-ray film (X-Omat, Eastman Kodak Co.) for 1–2 days. Confluent cultures of human iliac vein endothelial cells (HIVE) in 100-mm dishes were collected without trypsin and affinity-labeled as above. Nonspecific binding was determined by incubating HIVE with 7.5 nm cold TGFβ1 (a 50-fold excess) for 30 min before the addition of 125I-TGFβ1. The extent of phosphorylation of a protein kinase C (PKC)-specific peptide substrate by platelet lysates was measured using a kit supplied by Amersham Pharmacia Biotech. All procedures were performed on ice and according to the manufacturer's instructions. Equal numbers of platelets (3 × 107) in PRP were centrifuged, washed with calcium-free Tyrode's buffer, and sonicated in lysis buffer. 25 μl of lysate was added to a mixture containing reaction buffer, artificial membranes, 10 μm4-phorbol 12-myristate 13-acetate (PMA), peptide substrate, and [γ-32P]ATP. After 15 min, the reaction was stopped with acid and the peptide substrate was captured on binding paper, washed in acid, and counted in a scintillation counter. Controls included reactions lacking platelet lysates or the peptide substrate to determine background counts and endogenous PKC substrate levels, respectively. Scid Tgfb1 −/− mice exhibit outward signs of a mild bleeding disorder. Brains of 5/6Scid Tgfb1 −/− neonates exhibited multiple petechia indicative of microhemorrhages, while none of theScid Tgfb1 +/+ neonates examined (0/4) had this condition. Adult Scid Tgfb1 −/− mice routinely have “flushed” small bowels that show no signs of inflammation by histology. Furthermore, these mice tend to bleed more readily from incisions.Scid Tgfb1 −/− mice have bleeding times nearly twice as long as wild type litter-mates (TableI). The numbers of peripheral blood platelets in both Scid and non-Scid Tgfb1 −/− mice are elevated approximately 3-fold over those from Scid or non-Scid Tgfb1 +/+ and Scid or non-Scid Tgfb1+/− controls. Megakaryocyte counts from bone marrow are also significantly (p < 0.05) elevated in all Scid or non-Scid Tgfb1 −/− mice.Table IPlatelet counts, megakaryocyte counts, and bleeding timesPlatelet countMegakaryocyte count/10× fieldLateral tail stick bleeding time+/+, +/−−/−+/+, +/−−/−+/+, +/−−/−× 103/mm3sScid (C3H)188 ± 34578 ± 129*3.0 ± 0.55.4 ± 1.1*97.6 ± 4.4190 ± 5.0**129 BSw CF1163 ± 54550 ± 190**3.0 ± 0.95.0 ± 1.8***ND129 BSw CF1 mice are 129S2/SvPas × Black/Swiss × CF-1.Scid (C3H) refers to 129 BlSw CF1 mice backcrossed for three generations to a C3H strain homozygous for theScid locus. Blood platelet and bone marrow megakaryocyte counts obtained from 129 × CF-1 (8 pair) andScid (5 pair) mice are presented as means ± S.D. Lateral tail bleeding times were determined on the same day using three mice for each group and are reported as means ± S.D. Bleeding times from wild-type (non-Scid) inbred C3H mice and from non-Scid (Tgfb1 +/+) mice on the mixed C3H, 129, CF-1 background all had bleeding times which were within 10% of that of the Scid Tgfb1 +/+ control mice (not shown). +/+, +/−, and −/− indicate Tgfb1genotype. Results that are significantly different from control (+/+ and +/−) values are indicated as follows: p < 0.001; *, p< 0.01; **, p < 0.001; ***, p < 0.05. ND, not determined. Open table in a new tab 129 BSw CF1 mice are 129S2/SvPas × Black/Swiss × CF-1.Scid (C3H) refers to 129 BlSw CF1 mice backcrossed for three generations to a C3H strain homozygous for theScid locus. Blood platelet and bone marrow megakaryocyte counts obtained from 129 × CF-1 (8 pair) andScid (5 pair) mice are presented as means ± S.D. Lateral tail bleeding times were determined on the same day using three mice for each group and are reported as means ± S.D. Bleeding times from wild-type (non-Scid) inbred C3H mice and from non-Scid (Tgfb1 +/+) mice on the mixed C3H, 129, CF-1 background all had bleeding times which were within 10% of that of the Scid Tgfb1 +/+ control mice (not shown). +/+, +/−, and −/− indicate Tgfb1genotype. Results that are significantly different from control (+/+ and +/−) values are indicated as follows: p < 0.001; *, p< 0.01; **, p < 0.001; ***, p < 0.05. ND, not determined. To test for a deficiency in platelet aggregation, PRP collected from mice via venipuncture was assayed for aggregation competence. When stimulated with the agonists ADP, collagen, or the TXA2 analog I-BOP (28Dorn G.W. DeJesus A. Am. J. Physiol. 1991; 260: H327-H334Crossref PubMed Google Scholar), Scid Tgfb1 −/− platelets fail to properly aggregate as compared with Scid Tgfb1 +/+control platelets (Fig. 1). Similar results are obtained with ADP using platelets recovered fromRag2 −/− Tgfb1 −/−mice, which also have no inflammatory disease (data not shown).Scid Tgfb1 −/− platelets show a range of aggregation responses to 10 μm ADP from approximately 0–80% of controls. In contrast to ADP-mediated responses, Scid Tgfb1 −/− platelets consistently fail to aggregate properly following stimulation with either 20 μg/ml collagen or 1 μm I-BOP. TGFβ1-deficient platelets that show an 80% aggregation response to ADP fail to aggregate when stimulated with I-BOP (data not shown), indicating an agonist-specific aggregation response that is not due to variability in platelet collection. Unlike these other agonists, thrombin induces maximal, yet delayed, aggregation in platelets fromScid Tgfb1 −/− and Scid Tgfb1 +/+ mice (Fig. 1). Flow cytometry experiments using a fluorescent antibody to fibrinogen reveals that only 39 ± 25% (n = 7) of Scid Tgfb1 −/− platelets bind normal levels of fibrinogen after stimulation with ADP as compared with 75 ± 15% (n = 7) of platelets from littermate controls (Fig.2 A). In response to I-BOP, only 46 ± 9% (n = 3) of Scid Tgfb1 −/− platelets bind normal levels of fibrinogen compared with 75 ± 8% (n = 3) of control platelets. Similar numbers of Scid Tgfb1 −/− and control platelets bind normal levels of fibrinogen in response to thrombin (Fig. 2 B) (96 ± 3% versus 97 ± 2%, respectively), consistent with the ability of thrombin to induce full aggregation. The reduced fraction of Scid Tgfb1 −/−platelets that bound fibrinogen after stimulation with ADP reflects the fact that these platelets release fibrinogen faster thanScid Tgfb1 +/+ control platelets following ADP activation (Fig. 2 C). Normal fibrinogen binding in response to thrombin indicates normal levels of αIIb/β3 receptors on TGFβ1-deficient platelets, which was confirmed by flow cytometric measurement of αIIb/β3 receptor density. TGFβ1-deficient platelets exhibit β3-specific mean fluorescence/platelet of 59.1 ± 16.4 (n = 3) versus46.2 ± 8.6 (n = 4) for control platelets. Plasma from TGFβ1-deficient mice contains normal levels of calcium and fibrinogen and can support normal aggregation of wild type platelets in response to ADP (data not shown). Platelets from Scid Tgfb1 −/− mice exhibit a rapid shape change in response to agonists as detected by the drop in light transmission during in vitro aggregation to ADP, I-BOP, and collagen (Fig. 1). In addition, TGFβ1-deficient platelets produce normal levels of TXA2 and mobilize P-selectin to the platelet surface in response to ADP (Table II). P-selectin levels of non-stimulated platelets from both Scid Tgfb1 −/− and Tgfb1 +/+mice were similar to background levels, indicating that our collection protocol does not prestimulate the platelets (Table II). Stimulation ofScid Tgfb1 −/− platelets with PMA, which activates PKC independently of receptor-mediated upstream events, also results in delayed aggregation (Fig.3 A). However, when measured directly, total PKC activity in Scid Tgfb1 −/− platelets is not different from control platelets (Fig. 3 A), indicating that there is no reduction in PKC enzymes. Consequently, the defect in aggregation lies downstream of PKC in its activation cascade.Table IIProduction of thromboxane B2 and expression of P-selectin following ADP activationScid Tgfb1 +/+Scid Tgfb1 −/−TXB2aThromboxane B2 (TXB2) (pg/μl), a stable metabolite of TXA2, in plasma or supernatants of 1.6 × 107 platelets (in PRP) activated for 10 min at 37 °C with 10 μm ADP was detected by enzyme-linked immunosorbent assay. Measurements of plasma and platelet-derived TXB2 were made from the same three mice for each genotype. Plasma0.5 ± 0.120.6 ± 0.08 Supernatants1.3 ± 0.061.7 ± 0.35P-selectinbThe percentage of platelets positive for surface P-selectin staining before (resting) or following activation (activated) by ADP was measured by flow cytometry using a FITC-conjugate antibody directed against mouse P-selectin. Resting11.4 ± 1.84.2 ± 1.4 Activated22 ± 627 ± 4Values are means ± S.E. of three mice.a Thromboxane B2 (TXB2) (pg/μl), a stable metabolite of TXA2, in plasma or supernatants of 1.6 × 107 platelets (in PRP) activated for 10 min at 37 °C with 10 μm ADP was detected by enzyme-linked immunosorbent assay. Measurements of plasma and platelet-derived TXB2 were made from the same three mice for each genotype.b The percentage of platelets positive for surface P-selectin staining before (resting) or following activation (activated) by ADP was measured by flow cytometry using a FITC-conjugate antibody directed against mouse P-selectin. Open table in a new tab Values are means ± S.E. of three mice. To test if the presence of TGFβ1 affects platelet aggregation, Scid Tgfb1 −/−platelets were incubated with human recombinant TGFβ1 before and during ADP stimulation. Pretreatment of these platelets with 20 ng/ml TGFβ1 facilitates aggregation in response to ADP (Fig.3 B). The degree of rescue by TGFβ1 is independent of the extent of aggregation by TGFβ1-deficient platelets prior to addition of TGFβ1. Those platelets that initially fail to respond to ADP show the same absolute degree of response when preincubated with TGFβ1 as do platelets from Scid Tgfb1 −/−mice that exhibit 80% responsiveness to ADP alone (Fig.3 B). Co-incubation with a neutralizing antibody against TGFβ1 prevents the effect of added TGFβ1 on aggregation (data not shown). In order for the added TGFβ1 to facilitate aggregation, it is necessary to preincubate mutant platelets with at least 20 ng/ml TGFβ1 for 1 h or longer before inducing aggregation. Shorter incubation times or lower concentrations result in no improvements to aggregation. Furthermore, the addition of TGFβ1 to wild type platelets has no effect on aggregation responses to ADP, nor does TGFβ1 alone induce aggregation in either Scid Tgfb1 −/− or Scid Tgfb1+/+platelets (data not shown). The effect of exogenous TGFβ1 on Scid Tgfb1 −/− platelets demonstrates that TGFβ1 signals in platelets. To address whether the receptors are present to mediate TGFβ1 signaling, we examined mouse platelets for125I-TGFβ1 binding. Platelets from both Scid Tgfb1 +/+ and Scid Tgfb1 −/− mice bind 125I-TGFβ1 mainly through types I and II receptors (Fig.4). Human platelets also have the type II receptor (data not shown). In contrast to human iliac vein endothelial cells, murine platelets do not possess endoglin. Furthermore, murine platelets lack the type III receptor. Mice lacking TGFβ1 produce increased numbers of bone marrow megakaryocytes and circulating platelets. Previous studies have indicated that TGFβ1 inhibits bone marrow-derived megakaryocyte growth, colony formation, and platelet production (29Ishibashi T. Miller S.L. Burstein S.A. Blood. 1987; 69: 1737-1741Crossref PubMed Google Scholar, 30Kuter D.J. Gminski D.M. Rosenberg R.D. Blood. 1992; 79: 619-626Crossref PubMed Google Scholar, 31Zauli G. Vitale L. Brunelli M.A. Bagnara G.P. Exp. Hematol. 1992; 20: 850-854PubMed Google Scholar, 32Carlino J.A. Higley H.R. Creson J.R. Avis P.D. Ogawa Y. Ellingsworth L.R. Exp. Hematol. 1992; 20: 943-950PubMed Google Scholar). The results observed in both the Scid and non-Scid Tgfb1 −/− mice are consistent with this role for TGFβ1 as a negative regulator of megakaryocyte growth and hence platelet production. Additionally, these mice exhibit a mild bleeding disorder characterized by a 2-fold longer bleeding time, cerebral petechia in neonates, and a “flushed” appearing small bowel in adults. Associated with the elevated platelet numbers is an aggregation deficiency in response to ADP, collagen and I-BOP, a TXA2analog. Thrombin, a potent platelet agonist, induces slightly delayed but full aggregation. The platelet aggregation defect is sufficient to explain the bleeding disorder. However, contributing factors such as vascular fragility or endothelial cell dysfunction cannot be discounted. Essential thrombocythemia, a human myeloproliferative disorder, is characterized by clonal expansion of megakaryocytes, persistent elevated platelet counts, and abnormal platelet function (33Tobelem G. Baillieres Clin. Haematol. 1989; 2: 719-728Abstract Full Text PDF PubMed Scopus (29) Google Scholar, 34Wehmeier A. Sudhoff T. Meierkord F. Semin. Thromb. Hemost. 1997; 23: 391-402Crossref PubMed Scopus (76) Google Scholar). The similarities between TGFβ1-deficient mice and essential thrombocythemia patients suggest that defects in the TGFβ1 signaling pathway or a pathway regulated by TGFβ1 may be altered in essential thrombocythemia patients. Platelet aggregation in response to ADP is poor but variable. The cause of this variability is not clear. Platelets from Scid Tgfb1 −/− mice that responded well to ADP failed to aggregate in response to the thromboxane A2 analog, I-BOP, suggesting that the variability is specific to ADP. The difference in responses to ADP and I-BOP also indicates that, unlike human platelets, murine platelets do not depend on TXA2 as an intermediate in ADP-stimulated aggregation. Recently, murine platelets were shown to be insensitive to cyclooxygenase inhibitors (35Johnson E.N. Brass L.F. Funk C.D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3100-3105Crossref PubMed Scopus (107) Google Scholar), consistent with our observations. From this, we conclude that ADP activates mouse platelets through a direct, TXA2-independent pathway and that ADP is a stronger platelet agonist in mouse than in human. Thus, the variability in Scid Tgfb1 −/− platelet response to ADP may reflect the strong stimulation by ADP that can partially override the defect. Even though TGFβ1-deficient platelets do not aggregate properly in response to ADP, they do change shape and generate TXA2. Furthermore, TGFβ1-deficient platelets exhibit normal agonist-induced α-granule secretion. However, platelets from Scid Tgfb1 −/− mice exhibit impaired fibrinogen binding, which can fully account for the defective aggregation. These observations suggest that this defect occurs late in the activation cascade, specific to αIIb/β3 activation. Similar to Scid Tgfb1 −/− mouse platelets, normal human platelets treated with phosphoinositol 3-kinase (PI3-K) inhibitors change shape, produce thromboxane, and mobilize P-selectin in response to agonists. However, PI3-K-inhibited platelets are unable to induce and maintain αIIb/β3in an active form (36Kovacsovics T.J. Bachelot C. Toker A. Vlahos C.J. Duckworth B. Cantley L.C. Hartwig J.H. J. Biol. Chem. 1995; 270: 11358-11366Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 37Zhang J. Shattil S.J. Cunningham M.C. Rittenhouse S.E. J. Biol. Chem. 1996; 271: 6265-6272Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). The similarity strongly suggests the aggregation defect observed in Scid Tgfb1 −/− platelets reflects impaired PI3-K-mediated αIIb/β3 activation. Our results show that murine platelets bind TGFβ1 through a competent TGFβ type I/II receptor system. Binding of TGFβ1 to both receptor types requires receptor heterodimerization, thereby forming the active signaling complex (38Wrana J.L. Attisano L. Carcamo J. Zentella A. Doody J. Laiho M. Wang X.F. Massague J. Cell. 1992; 71: 1003-1014Abstract Full Text PDF PubMed Scopus (1372) Google Scholar). In murine platelets binding of125I-TGFβ1 to both types I and II receptors indicates that heterodimerization is occurring. Unlike endothelial cells, which share a common lineage with platelets, murine platelets do not bind TGFβ1 through endoglin (Fig. 4). Furthermore, platelets do not posses the type III binding protein. Thus, any TGFβ1-derived signals would be transduced from primarily the classic type I/II receptor system. Because platelets lack a nucleus, TGFβ1 must be regulating platelet activity independent of transcription. In other cell types the type II TGFβ receptor can interact with the α subunit of farnesyltransferase (18Kawabata M. Imamura T. Miyazono K. Engel M.E. Moses H.L. J. Biol. Chem. 1995; 270: 29628-29631Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 39Wang T. Danielson P.D. Li B.Y. Shah P.C. Kim S.D. Donahoe P.K. Science. 1996; 271: 1120-1122Crossref PubMed Scopus (107) Google Scholar), suggesting that TGFβ1 may be affecting protein prenylation states in platelets. In addition, TGFβ1 can regulate protein translation and stability (19Sandhu C. Garbe J. Bhattacharya N. Daksis J. Pan C.H. Yaswen P. Koh J. Slingerland J.M. Stampfer M.R. Mol. Cell. Biol. 1997; 17: 2458-2467Crossref PubMed Google Scholar, 40Finder J. Stark W.W.J. Nakayama D.K. Geller D. Wasserloos K. Pitt B.R. Davies P. Am. J. Physiol. 1995; 268: L862-L867PubMed Google Scholar) and may play a similar role in platelets. The length of preincubation required for TGFβ1 to facilitate platelet function is consistent with either of these two mechanisms. Preliminary studies using puromycin suggest that TGFβ1 does not signal through protein translation (data not shown). Further studies involving TGFβ1-deficient mouse platelets should prove useful in identifying non-nuclear mediators of TGFβ signaling and may reveal additional insights into platelet activation mechanisms. Preincubating platelets from Scid Tgfb1 −/− mice with active TGFβ1 facilitates aggregation. However, TGFβ1 alone does not induce aggregation. Thus TGFβ1 augments rather than initiates aggregation. The time of TGFβ1 preincubation (>1 h) needed to affect platelet activity and the relatively short time of ADP aggregation (5 min) suggest that TGFβ1 is not acting during platelet aggregation. Instead, it may precondition the platelet to ensure a vigorous response in conditions requiring rapid and complete formation of a hemostatic plug. In this regard, the long preincubation period may mimic the continual exposure to low levels of TGFβ1 during the life of the platelet in the wild type mouse. This may also explain why platelets from control mice are unresponsive to exogenous TGFβ1. Plasma contains low levels of circulating, active TGFβ1 bound to carriers such as α-macroglobulin (41Kropf J. Schurek J.O. Wollner A. Gressner A.M. Clin. Chem. 1997; 43: 1965-1974Crossref PubMed Scopus (124) Google Scholar, 42Webb D.J. Wen J. Lysiak J.J. Umans L. Van Leuven F. Gonias S.L. J. Biol. Chem. 1996; 271: 24982-24988Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). The presence of the TGFβ1 carriers in the plasma ofScid Tgfb1 −/− mice may account for why a relatively large amount of TGFβ1 (20 ng/ml) was needed to improve aggregation responses of Scid Tgfb1 −/− platelets. Presumably, these carrier molecules are depleted of TGFβ1 in the Scid Tgfb1 −/− mice and may sequester a significant portion of the exogenously added active TGFβ1 during our experiments. In conclusion, the absence of TGFβ1 in mice leads to compromised platelet aggregation resulting in a mild bleeding disorder. The inability of TGFβ1-deficient platelets to aggregate properly is due to faulty fibrinogen binding involving downstream targets of PKC (e.g. PI3-K). The compromise in platelet activity within theScid Tgfb1 −/− mouse demonstrates that TGFβ1 is important in hemostatic activities other than putative vascular cell regulation. Because platelets lack a nucleus, such activities would be independent of nuclear-mediated responses such as gene transcription. We thank Wen Yun Sun for expert assistance in collecting blood and PCR genotyping; Sandra J. Engle for intellectual input into these experiments; and Maureen Luehrmann, Bing Tao Fan, Colleen York, and Christy Sloan for animal husbandry. Human iliac vein endothelial cells were kindly provided by Dr. Stuart K. Williams (University of Arizona)." @default.
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• W1990595551 title "Transforming Growth Factor β1 Enhances Platelet Aggregation through a Non-transcriptional Effect on the Fibrinogen Receptor" @default.
• W1990595551 cites W1506960040 @default.
• W1990595551 cites W1524114372 @default.
• W1990595551 cites W1550778478 @default.
• W1990595551 cites W1569456617 @default.
• W1990595551 cites W1601529928 @default.
• W1990595551 cites W1634736267 @default.
• W1990595551 cites W1663371441 @default.
• W1990595551 cites W1971111760 @default.
• W1990595551 cites W1975148930 @default.
• W1990595551 cites W1999340769 @default.
• W1990595551 cites W1999397857 @default.
• W1990595551 cites W2003661065 @default.
• W1990595551 cites W2004589420 @default.
• W1990595551 cites W2014919830 @default.
• W1990595551 cites W2021263701 @default.
• W1990595551 cites W2039892496 @default.
• W1990595551 cites W2044069347 @default.
• W1990595551 cites W2062353230 @default.
• W1990595551 cites W2065993385 @default.
• W1990595551 cites W2066508034 @default.
• W1990595551 cites W2070758335 @default.
• W1990595551 cites W2079517510 @default.
• W1990595551 cites W2093535914 @default.
• W1990595551 cites W2102868284 @default.
• W1990595551 cites W2116591510 @default.
• W1990595551 cites W2117811396 @default.
• W1990595551 cites W2128048856 @default.
• W1990595551 cites W2134524472 @default.
• W1990595551 cites W2142250806 @default.
• W1990595551 cites W2159141578 @default.
• W1990595551 cites W2164615751 @default.
• W1990595551 cites W2276591666 @default.
• W1990595551 cites W2341344091 @default.
• W1990595551 cites W33813833 @default.
• W1990595551 cites W4231423149 @default.
• W1990595551 cites W4235438234 @default.
• W1990595551 cites W4323241890 @default.
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