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J Biol Chem, Vol. 274, Issue 43, 31008-31013, October 22, 1999

Transforming Growth Factor 1 Enhances Platelet Aggregation through a Non-transcriptional Effect on the Fibrinogen Receptor^*

James B. Hoying^§, Moying Yin, Ronald Diebold^¶, Ilona Ormsby, Ann Becker, and Thomas Doetschman^**

From the  Program of Excellence in Molecular Biology, Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati and the  Division of Hematology, Cincinnati Children's Hospital, Cincinnati, Ohio 45267-0524

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Upon activation, platelets store and release large amounts of the peptide transforming growth factor 1 (TGF1). The released TGF1 can then act on nearby vascular cells to mediate subsequent vessel repair. In addition, TGF1 may circulate to bone marrow and regulate megakaryocyte activity. It is not known what effect, if any, TGF1 has on platelets. Adult TGF1-deficient mice exhibit thrombocythemia and a mild bleeding disorder that is shown to result from faulty platelet aggregation. TGF1-deficient platelets are shown to contain functional receptors, and preincubation with recombinant TGF1 improves aggregation, demonstrating that TGF1 plays an active role in platelet aggregation. TGF1-deficient platelets fail to retain bound fibrinogen in response to aggregation agonists, but they possess normal levels of the _IIb/₃ 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 _IIb/₃ into a fibrinogen-binding conformation is impaired in the absence of TGF1. 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 TGF1 signaling pathway that enhances the activation and maintenance of integrin function.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 (1, 2). Storage granules are then mobilized to the platelet surface, releasing a variety of molecules that mediate subsequent hemostatic events (3). TGF1¹ is stored in large amounts as a latent peptide in the secretory -granules of circulating platelets and is one of the molecules released during platelet activation (4). Active platelets can release enough TGF1 to raise the local concentration of TGF1 to as much as 40 ng/ml at the injury site and in the developing thrombus (5). TGF1 can influence vessel repair through regulation of endothelial cell function (6, 7), smooth muscle cell differentiation (8, 9), and vessel wall remodeling (10, 11). It is not known whether TGF1 can also regulate platelet activity.

In nucleated cells, members of the Smad gene family are believed the primary mediators of intracellular signaling from the TGF receptor types I and II (12, 13). SMADs are able to bind DNA (14, 15) and can regulate transcription of TGF1-responsive genes (16, 17). However, the type I receptor can interact with other molecules such as the  subunit of farnesyltransferase (18). Additionally, TGF1 can stabilize protein levels in epithelial cells (19). These findings suggest that there are possible transcription-independent signaling pathways for TGF1 (19).

The primary defect in the TGF1-deficient mouse is a severe multifocal inflammatory disease resulting in death by the 3rd week after birth (20-22). Genetic combination of Tgfb1 knockout and Scid alleles eliminates the inflammation and increases longevity by 3-5 months (23), thereby permitting investigation of adult phenotypes primary to the absence of TGF1 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 TGF1 improves platelet aggregation, indicating the presence of a TGF1 signaling pathway in platelets. Because TGF1 signaling in platelets is necessarily independent of transcription, these results demonstrate the existence of a non-transcriptional TGF1 signaling pathway that mediates platelet function by affecting the state of integrin activation.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Mice-- Tgfb1^/ mice are on a mixed strain background of 129S2/SvPas × BL/Swiss × CF-1 (approximately 25%, 25%, and 50%, respectively) (20). To place the null Tgfb1 allele on an immune-compromised background, they were back-crossed for three generations to C3H mice homozygous for the Scid allele (23). For all strains, experimental animals were obtained through Scid Tgfb1^+/ × Scid Tgfb1^+/ matings. When possible, Scid Tgfb1^+/+ and Scid Tgfb1^+/ siblings of Scid Tgfb1^/ offspring were used as controls.

Platelet and Megakaryocyte Counts-- 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 Time-- Bleeding times were determined by a previously reported method (24). 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.

Platelet Aggregation-- 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 from Scid 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 mM glycyl-prolyl-arginyl-proline peptide (GPRP, Sigma), to prevent fibrin clot formation (25). For experiments involving preincubation with TGF1, 250 µl of PRP was incubated at room temperature for at least 1 h with active recombinant human TGF1 (R&D Systems) prior to performing the aggregation experiment.

Fibrinogen Binding and Flow Cytometry-- Detection of bound fibrinogen was carried out essentially as described previously (25). 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 mM MgSO₄, 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-[1,2(5Z),3(1E,3R*),4])- 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 _IIb/₃ (26) did not interact with the mouse platelets (from either Tgfb1^+/+ or Tgfb1^/ mice) used in our experiments.

Measurement of Thromboxane Production-- 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.

Affinity Cross-linking of ¹²⁵I-TGF1 to Receptors-- Identification of platelet surface TGF1-binding proteins was performed by affinity cross-linking of ¹²⁵I-TGF1 with disuccinimidyl suberate (27). 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 pM ¹²⁵I-TGF1 in 0.5 ml of binding buffer. All incubations were performed in siliconized microcentrifuge tubes to minimize binding of the ¹²⁵I-TGF1 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 mM EDTA, 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 TGF1 (a 50-fold excess) for 30 min before the addition of ¹²⁵I-TGF1.

Protein Kinase C Activity-- 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 µM 4-phorbol 12-myristate 13-acetate (PMA), peptide substrate, and [-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.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

ScidTgfb1^/ Mice Exhibit a Mild Bleeding Disorder and Elevated Circulating Platelet Numbers-- Scid Tgfb1^/ mice exhibit outward signs of a mild bleeding disorder. Brains of 5/6 Scid Tgfb1^/ neonates exhibited multiple petechia indicative of microhemorrhages, while none of the Scid 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 (Table I). 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.

View this table: [in this window] [in a new window]   Table I Platelet counts, megakaryocyte counts, and bleeding times 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 the Scid locus. Blood platelet and bone marrow megakaryocyte counts obtained from 129 × CF-1 (8 pair) and Scid (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 Tgfb1 genotype. 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.

Scid Tgfb1^/ Platelets Do Not Aggregate Properly in Vitro-- 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 (28), 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 from Rag2^/ 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. TGF1-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 from Scid Tgfb1^/ and Scid Tgfb1^+/+ mice (Fig. 1).

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Aggregation of platelets from Scid Tgfb1+/+ and Tgfb1/ mice in response to various agonists. A, PRP is stirred at 37 °C in a photo-aggregometer and the resulting aggregation following agonist addition is measured as the percentage of light transmission through a platelet-poor plasma blank. The agonist was added at "0" min and the aggregation response followed until the traces reached a plateau. Changes in platelet shape immediately following agonist addition appear as a drop in the percent of transmitted light (arrow in traces for ADP, collagen, and I-BOP). Each aggregation is performed with PRP pooled from three to five mice and is representative of at least four separate experiments. The traces shown for ADP labeled 1-3 are of three separate aggregation experiments using pooled platelets from different groups of TGF1-deficient mice and show the possible range of responses obtained with this agonist. The concentration of ADP, I-BOP, collagen, and thrombin used in the aggregations was previously determined to produce a maximal response in wild type mouse platelets. For aggregation with thrombin, 0.25 mM GPRP peptide, which inhibits fibrinogen clot formation, was included. B, distribution of aggregation responses to ADP (10 µM) by platelets from Scid Tgfb1+/+ () and Scid Tgfb1/ () mice. Each data point represents an aggregation experiment involving platelets pooled from three to five mice. Each aggregation experiment with Tgfb1/ platelets was accompanied by a separate experiment with wild type mouse platelets using the same reagents. Paired Scid Tgfb1+/+ and Scid Tgfb1/ aggregation experiments are plotted together at the same x axis position. In response to ADP, TGF1-deficient platelets from 13/16 (81%) experiments aggregated to 20% of maximum or less.

Scid Tgfb1^/ Platelets Improperly Bind Fibrinogen-- 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. 2A). 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. 2B) (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 than Scid Tgfb1^+/+ control platelets following ADP activation (Fig. 2C). Normal fibrinogen binding in response to thrombin indicates normal levels of _IIb/₃ receptors on TGF1-deficient platelets, which was confirmed by flow cytometric measurement of _IIb/₃ receptor density. TGF1-deficient platelets exhibit ₃-specific mean fluorescence/platelet of 59.1 ± 16.4 (n = 3) versus 46.2 ± 8.6 (n = 4) for control platelets. Plasma from TGF1-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).

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Flow cytometry of fibrinogen binding to Scid Tgfb1+/+ and Scid Tgfb1/ mouse platelets following stimulation. Fibrinogen binding was detected with a FITC-anti human fibrinogen antibody following stimulation with 10 µM ADP (A) or 10 units/ml thrombin (B). Representative traces are shown. In A, fluorescence traces of platelets in the absence (ADP) and presence (+ADP) of ADP are shown superimposed on one another. Panel B depicts only those traces obtained following thrombin stimulation. The level of fluorescence defined by the M1 region in each panel was used to determine the percentage of platelets positive for normal fibrinogen binding (see text). C, time course of IIb/3 inactivation following stimulation with ADP. The FITC-conjugated antibody against fibrinogen was added at the indicated times after stimulation with 10 µM ADP. Thirty minutes after the addition of antibody, samples were fixed in formalin and analyzed by flow cytometry. Results are presented as the percentage of platelets positive for bound fibrinogen determined at the time 0 point.

Delayed Aggregation following PKC Activation of Scid Tgfb1^/ Platelets-- 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, TGF1-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 of Scid Tgfb1^/ platelets with PMA, which activates PKC independently of receptor-mediated upstream events, also results in delayed aggregation (Fig. 3A). However, when measured directly, total PKC activity in Scid Tgfb1^/ platelets is not different from control platelets (Fig. 3A), indicating that there is no reduction in PKC enzymes. Consequently, the defect in aggregation lies downstream of PKC in its activation cascade.

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Effect of TGF1 on aggregation of TGF1-deficient platelets. A, a representative trace of PMA-induced platelet aggregation. Pooled PRP from Scid Tgfb1+/+ (+/+) or Scid Tgfb1/ (/) mice was stimulated by 10 µM PMA with stirring in an aggregometer. PMA was added at time 0 and the change in light transmission followed for 25 min. Inset, total PKC activity in lysates from equal numbers of platelets from Scid Tgfb1+/+ and Scid Tgfb1/ mice. Values are mean ± S.E. of (n) mice indicating the picomoles of Pi/min/3 × 107 platelets. Results are corrected for the labeling of endogenous PKC substrates. B, improved aggregation of platelets in the presence of TGF1. Representative aggregation tracings from two separate experiments (1 and 2) similar to those described in Fig. 1. PRP pooled from four TGF1-deficient mice were prepared for aggregation with PPP as described in Fig. 1 and incubated at room temperature with (+TGF1) or without (TGF1) recombinant human TGF1 (20 ng/ml) for 1 h before activation with 10 µM ADP in the aggregometer. Incubated samples were used directly for aggregation so that recombinant TGF1 was present during the aggregation. An aggregation trace of platelets in plasma from Scid Tgfb1+/+ mice (+/+) incubated at room temperature for 1 h is included as a reference.

Addition of Active TGF1 to TGF1-deficient Platelets Improves Defective Aggregation-- To test if the presence of TGF1 affects platelet aggregation, Scid Tgfb1^/ platelets were incubated with human recombinant TGF1 before and during ADP stimulation. Pretreatment of these platelets with 20 ng/ml TGF1 facilitates aggregation in response to ADP (Fig. 3B). The degree of rescue by TGF1 is independent of the extent of aggregation by TGF1-deficient platelets prior to addition of TGF1. Those platelets that initially fail to respond to ADP show the same absolute degree of response when preincubated with TGF1 as do platelets from Scid Tgfb1^/ mice that exhibit 80% responsiveness to ADP alone (Fig. 3B). Co-incubation with a neutralizing antibody against TGF1 prevents the effect of added TGF1 on aggregation (data not shown). In order for the added TGF1 to facilitate aggregation, it is necessary to preincubate mutant platelets with at least 20 ng/ml TGF1 for 1 h or longer before inducing aggregation. Shorter incubation times or lower concentrations result in no improvements to aggregation. Furthermore, the addition of TGF1 to wild type platelets has no effect on aggregation responses to ADP, nor does TGF1 alone induce aggregation in either Scid Tgfb1^/ or Scid Tgfb1^+/+ platelets (data not shown).

Murine Platelets Possess Type I and II TGF1 Receptors-- The effect of exogenous TGF1 on Scid Tgfb1^/ platelets demonstrates that TGF1 signals in platelets. To address whether the receptors are present to mediate TGF1 signaling, we examined mouse platelets for ¹²⁵I-TGF1 binding. Platelets from both Scid Tgfb1^+/+ and Scid Tgfb1^/ mice bind ¹²⁵I-TGF1 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.

View larger version (105K): [in this window] [in a new window]   Fig. 4.   Autoradiograph of 125I-TGF1 cross-linked platelets detecting TGF1-binding proteins on the platelet surface. Equal numbers of platelets from Scid Tgfb1+/+ mice (+/+) and Scid Tgfb1/ mice (/) were washed twice in the presence of 1 µM prostacyclin and labeled by chemical cross-linking to 125I-TGF1. Platelet lysates were separated on 7% polyacrylamide gel electrophoresis gel under reducing (R) or nonreducing (NR) conditions and the position of receptor-bound 125I-TGF1 was detected by exposure to film. Under reducing conditions, the 185 kDa endoglin molecule migrates to a position near the type II receptor while under non-reducing conditions, endoglin migrates as a broad smear at the top of the gel. We detected no differences in TGF1 receptor levels between platelets of Scid Tgfb1+/+ and Scid Tgfb1/ mice in three separate experiments. Experiments with HIVE, which possess endoglin but not type III receptor, were included for comparison. The result from preincubation of HIVE for 30 min with a 50-fold excess of cold TGF1 is shown in the rightmost lane under HIVE. The numbers to the right of the autoradiograph represent the positions of molecular markers in kDa. TRI, TGF receptor type I; TRII, TGF receptor type II.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Mice lacking TGF1 produce increased numbers of bone marrow megakaryocytes and circulating platelets. Previous studies have indicated that TGF1 inhibits bone marrow-derived megakaryocyte growth, colony formation, and platelet production (29-32). The results observed in both the Scid and non-Scid Tgfb1^/ mice are consistent with this role for TGF1 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 TXA₂ analog. 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 (33, 34). The similarities between TGF1-deficient mice and essential thrombocythemia patients suggest that defects in the TGF1 signaling pathway or a pathway regulated by TGF1 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 (35), 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 TGF1-deficient platelets do not aggregate properly in response to ADP, they do change shape and generate TXA₂. Furthermore, TGF1-deficient platelets exhibit normal agonist-induced -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 _IIb/₃ 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 _IIb/₃ in an active form (36, 37). The similarity strongly suggests the aggregation defect observed in Scid Tgfb1^/ platelets reflects impaired PI3-K-mediated _IIb/₃ activation.

Our results show that murine platelets bind TGF1 through a competent TGF type I/II receptor system. Binding of TGF1 to both receptor types requires receptor heterodimerization, thereby forming the active signaling complex (38). In murine platelets binding of ¹²⁵I-TGF1 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 TGF1 through endoglin (Fig. 4). Furthermore, platelets do not posses the type III binding protein. Thus, any TGF1-derived signals would be transduced from primarily the classic type I/II receptor system. Because platelets lack a nucleus, TGF1 must be regulating platelet activity independent of transcription. In other cell types the type II TGF receptor can interact with the  subunit of farnesyltransferase (18, 39), suggesting that TGF1 may be affecting protein prenylation states in platelets. In addition, TGF1 can regulate protein translation and stability (19, 40) and may play a similar role in platelets. The length of preincubation required for TGF1 to facilitate platelet function is consistent with either of these two mechanisms. Preliminary studies using puromycin suggest that TGF1 does not signal through protein translation (data not shown). Further studies involving TGF1-deficient mouse platelets should prove useful in identifying non-nuclear mediators of TGF signaling and may reveal additional insights into platelet activation mechanisms.

Preincubating platelets from Scid Tgfb1^/ mice with active TGF1 facilitates aggregation. However, TGF1 alone does not induce aggregation. Thus TGF1 augments rather than initiates aggregation. The time of TGF1 preincubation (>1 h) needed to affect platelet activity and the relatively short time of ADP aggregation (5 min) suggest that TGF1 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 TGF1 during the life of the platelet in the wild type mouse. This may also explain why platelets from control mice are unresponsive to exogenous TGF1. Plasma contains low levels of circulating, active TGF1 bound to carriers such as -macroglobulin (41, 42). The presence of the TGF1 carriers in the plasma of Scid Tgfb1^/ mice may account for why a relatively large amount of TGF1 (20 ng/ml) was needed to improve aggregation responses of Scid Tgfb1^/ platelets. Presumably, these carrier molecules are depleted of TGF1 in the Scid Tgfb1^/ mice and may sequester a significant portion of the exogenously added active TGF1 during our experiments.

In conclusion, the absence of TGF1 in mice leads to compromised platelet aggregation resulting in a mild bleeding disorder. The inability of TGF1-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 the Scid Tgfb1^/ mouse demonstrates that TGF1 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.

	ACKNOWLEDGEMENTS

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).

	FOOTNOTES

^* This work was funded by National Institutes of Health NICHD Grant HD26471, NHLBI Grant HL41496, and NIEHS Grant ES05652 (to T. D.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ Current address: Biomedical Engineering Program, University of Arizona, Tucson, AZ 85724.

^¶ Current address: Howard Hughes Medical Institute, University of Michigan Medical School, Ann Arbor, MI 48109-0650.

^** To whom correspondence and reprint requests should be addressed: Dept. of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, 231 Bethesda Ave., ML 524, Cincinnati, OH 45267-0524. Tel: 513-558-0090; Fax: 513-558-1885; E-mail: thomas.doetschman@uc.edu.

	ABBREVIATIONS

The abbreviations used are: TGF1, transforming growth factor 1 protein; Tgfb1, transforming growth factor 1 gene; Scid, severe combined immunodeficiency; TXA₂, thromboxane A₂; I-BOP, (1S-[1,2(5Z),3(1E,3R*),4])-7-(3-[3-hydroxy-4-(4'-iodophenoxy)-1-butenyl]-7-oxabicyclo[2.2.1]heptan-2-yl)-5-heptenoic acid; PRP, platelet-rich plasma; PPP, platelet-poor plasma; PMA, 4-phorbol 12-myristate 13-acetate; PKC, protein kinase C; PI3-K, phosphoinositol 3-kinase; FITC, fluorescein isothiocyanate; HIVE, human iliac vein endothelial cells.

	REFERENCES

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