Inorganic phosphate induces mammalian growth plate chondrocyte apoptosis in a mitochondrial pathway involving nitric oxide and JNK MAP kinase.

Chondrocytes in the hypertrophic zone of the growth plate undergo apoptosis during endochondral bone development via mechanisms that involve inorganic phosphate (Pi) and nitric oxide (NO). Recent evidence suggests that Pi-dependent NO production plays a role in apoptosis of cells in the resting zone as well. This study examined the mechanism by which Pi induces NO production and the signaling pathways by which NO mediates its effects on apoptosis in these cells. Pi decreased the number of viable cells based on MTT activity; the number of TUNEL-positive cells and the level of DNA fragmentation were increased, indicating an increase in apoptosis. Blocking NO production using the NO synthase (NOS) inhibitor L: -NAME or cells from eNOS(-/-) mice blocked Pi-induced chondrocyte apoptosis, indicating that NO production is necessary. NO donors NOC-18 and SNOG both induced chondrocyte apoptosis. SNOG also upregulated p53 expression, the Bax/Bcl-2 expression ratio, and cytochrome c release from mitochondria, as well as caspase-3 activity, indicating that NO induces apoptosis via a mitochondrial pathway. Inhibition of JNK, but not of p38 or ERK1/2, MAP kinase was able to block NO-induced apoptosis, indicating that JNK is necessary in this pathway. Pi elevates NO production via eNOS in resting zone chondrocytes, which leads to a mitochondrial apoptosis pathway dependent on JNK.

17ß-Estradiol regulates rat growth plate chondrocyte apoptosis through a mitochondrial pathway not involving nitric oxide or MAPKs.

Estrogens cause growth plate closure in both males and females, by decreasing proliferation and inducing apoptosis of postproliferative growth plate chondrocytes. In vitro studies using 17ß-estradiol (E(2)) conjugated to bovine serum albumin (E(2)-BSA) show that rat costochondral growth plate resting zone chondrocytes also respond to E(2). Moreover, they are regulated by E(2)-BSA via a protein kinase C and ERK MAPK signaling pathway that is functional only in female cells. To better understand how E(2) regulates apoptosis of growth plate chondrocytes, rat resting zone chondrocytes cells were treated with E(2) or E(2)-BSA. E(2) caused apoptosis in male and female resting zone and growth zone chondrocytes in a dose-dependent manner, based on elevated DNA fragmentation, terminal deoxynucleotidyl transferase dUTP nick end labeling staining and caspase-3 activation. E(2) also up-regulated p53 and Bax protein (Bcl-2-associated X protein) levels and induced release of cytochrome C from the mitochondria, indicating a mitochondrial apoptotic pathway. The apoptotic effect of E(2) did not involve elevated nitric oxide production or MAPKs. It was reduced by ICI 182780, which is an estrogen receptor (ER) antagonist and blocked by antibodies to Erα36, a membrane-associated ER. E(2)-BSA reduced cell viability and increased caspase-3 activity; ICI 182780 had no effect, but anti-ERα36 antibodies blocked the effect. The results indicate that estrogen is able to directly affect the cell population kinetics of growth plate chondrocytes by regulating apoptosis, as well as proliferation and differentiation in both resting zone and growth zone cells. They also have provided further information about the physiological functions of estrogen on longitudinal bone growth.

Thrombin peptide TP508 prevents nitric oxide mediated apoptosis in chondrocytes in the endochondral developmental pathway.

TP508 is a 23-amino acid peptide derived from human prothrombin that increases cartilage matrix production and reduces alkaline phosphatase activity without changing chondrocyte proliferation. This study tested the hypothesis that TP508 acts by blocking the onset of apoptosis associated with hypertrophy. Rat costochondral resting zone chondrocytes and human auricular chondrocytes were cultured in DMEM containing 50microM ascorbic acid and 10% FBS. Apoptosis was induced by treatment of confluent cultures with chelerythrine, tamoxifen, or inorganic phosphate (Pi) for 24h. One half of the cultures received TP508 (0, 0.7, or 7microg/ml). Apoptosis was assessed as a function of DNA fragmentation ([3H]-thymidine labeled DNA fragments), TUNEL staining, and cell viability using the MTT assay, as well as by assessing the Bcl-2/Bax mRNA and protein ratios and caspase-3 activity. The universal NO synthase inhibitor l-NMMA was used to assess the effect of NO production on chondrocyte apoptosis and specific NO synthase subspecies were identified using iNOS inhibitor 1400W and nNOS inhibitor vinyl-l-NIO, as well as l-NAME, which inhibits both iNOS and eNOS. Finally, we assessed if TP508 would block NO production induced by the apoptogens. Chelerythrine, tamoxifen and Pi-induced apoptosis and this was reversed by TP508. All apoptogens increased NO production and this was reduced by TP508. TP508 reduced NO levels to the same extent as 1400W but not to the same extent as l-NAME, suggesting that its effects are mediated primarily by iNOS. In addition, TP508 reduced the effect of chelerythrine to the same extent as 1400W and l-NAME, again indicating that it acts via inhibition of an iNOS pathway. TP508 also regulated Bcl-2/Bax mRNA in a time and dose-dependent manner. The Bcl-2/Bax mRNA ratio was 0.11 in the absence of TP508 at 1h and 4.95 at 7microg/ml TP508; by 3h the ratio was approximately 1 in both groups. The Bcl-2/Bax protein ratio also increased by 63% at 1h. TP508 did not affect caspase-3 activity. TP508 also caused a dose-dependent increase in protein kinase C (PKC) activity within 9min that was maximal at 270min. These results show that TP508 prevents apoptosis in growth plate chondrocytes via inhibition of iNOS-dependent NO and suggest a possible role for PKC in the mechanism.

Thrombin peptide (TP508) treatment of rat growth plate cartilage cells promotes proliferation and retention of the chondrocytic phenotype while blocking terminal endochondral differentiation.

A synthetic peptide representing the receptor-binding domain of human thrombin (TP508, also known as Chrysalin) accelerates fracture repair in rats via endochondral ossification and promotes repair of rabbit cartilage defects. To understand how this peptide might stimulate cartilage and bone formation, we employed an established in vitro model of growth plate cartilage regulation. Rat costochondral cartilage resting zone and growth zone chondrocytes were treated with 0, 0.07, 0.7, or 7 microg/ml TP508 or a scrambled peptide, TP508-SP. Proliferation ([3H]-thymidine incorporation) was examined in pre-confluent cultures; effects on cell number, alkaline phosphatase activity, [35S]-sulfate incorporation, and responsiveness to vitamin D metabolites were tested using confluent cultures. TP508 did not affect proliferation of resting zone cells but it caused a dose-dependent increase in cell number and DNA synthesis of growth zone cells. Alkaline phosphatase specific activity of resting zone cells was reduced by TP508, whereas [35S]-sulfate incorporation was increased. Neither parameter was affected in growth zone cell cultures. TP508 treatment for 24 h did not induce resting zone cells to respond to 1alpha,25(OH)2D3, either with respect to alkaline phosphatase activity or proteoglycan production. In contrast, TP508 treatment reduced the stimulatory effect of 24R,25(OH)2D3 on alkaline phosphatase but it did not alter the stimulatory effect of 24R,25(OH)2D3 on [35S]-sulfate incorporation. In cultures treated for 48, 72, or 140 h with TP508, 1alpha,25(OH)2D3 restored alkaline phosphatase activity to control levels but did not stimulate activity over levels observed in untreated control cultures. The stimulatory effect of TP508 on [35S]-sulfate incorporation was evident up to 48 h post-confluence but at later time points, proteoglycan production was comparable to that seen in control cultures, control cultures challenged with 1alpha,25(OH)2D3, and cultures treated with TP508 followed by 1alpha,25(OH)2D3. TP508-SP had no effect on any of the parameters tested. These results indicate that TP508 exerts maturation specific effects on chondrocytes in the endochondral lineage, promoting cartilage extracellular matrix synthesis over endochondral differentiation in resting zone cells and proliferation over differentiation of growth zone cells.

Acceleration of full-thickness wound healing in normal rats by the synthetic thrombin peptide, TP508.

Thrombin is an essential factor in hemostasis, inflammation, and tissue repair. The synthetic thrombin peptide, TP508, binds to high-affinity thrombin receptors and mimics cellular effects of thrombin at sites of tissue injury. Treatment of full-thickness excisional wounds in normal rats with a single topical application of 0.1 microg TP508 (14 pmol/cm2) reproducibly accelerates wound closure, yielding wounds that on average close 39% more than controls by day 7 (p < 0.001). Wounds treated with 1.0 microg TP508 are 35% and 43% (p < 0.001) smaller than controls on day 7 and 10, respectively. The early rate of closure is approximately 40% greater in TP508-treated than vehicle-treated wounds (20 versus 14 mm2/day) and remains higher through day 7. Breaking strength after closure is slightly greater (15-23%) in wounds treated with TP508 than with saline alone. Histologic comparisons show that TP508 enhances recruitment of inflammatory cells to the wound site within 24 hours post-injury. TP508 treatment also augments revascularization of injured tissue, as evidenced at day 7 by the larger size of functional vessels in the granulation tissue and by the directed development of blood vessels to wounds. These studies raise the possibility that TP508 may be clinically useful in management of open wounds.

Thrombin peptide, TP508, stimulates angiogenic responses in animal models of dermal wound healing, in chick chorioallantoic membranes, and in cultured human aortic and microvascular endothelial cells.

The alpha-thrombin peptide, TP508, accelerates the healing of full-thickness wounds in both normal and ischemic skin. In wounds treated with TP508, a pattern of increased vascularization is consistently observed both grossly and microscopically when compared to wounds treated with saline. One possible mechanism by which the peptide accelerates wound healing is by promoting revascularization of granulation tissue at the injured site. To evaluate the angiogenic potential of TP508, the peptide was tested in the chick embryo chorioallantoic membrane (CAM), where it increased the density and size of CAM blood vessels relative to controls. Additionally, TP508 stimulated chemokinesis and chemotaxis in a dose-dependent fashion in cultured human aortic and human microvascular endothelial cells. Taken together, these in vivo and in vitro data support an angiogenic role for TP508 in wound healing. A working model is presented to explain how this 23-amino-acid peptide, which lacks proteolytic activity, is generated during wound healing and contributes to the nonproteolytic functions associated with alpha-thrombin during tissue repair.

Thrombin regulates the expression of proangiogenic cytokines via proteolytic activation of protease-activated receptor-1.

In addition to its central role in blood coagulation and hemostasis, human alpha-thrombin is a growth factor for a variety of cell types, including monocytes and endothelial cells, involved in the control of angiogenesis. Different cytokines produced by mononuclear cells have been implicated in angiogenic processes associated with tissue repair and certain human malignancies. We have previously shown that thrombin enhances proliferative responses in T lymphocytes. More recently, we reported that interferon-gamma-differentiated monocytes have increased expression of protease-activated receptor-1 (PAR-1) and increased thrombin binding. Since cytokines may be involved directly and indirectly in angiogenesis, we initiated studies to determine thrombin effects on the induction of cytokines, such as interleukin (IL)-1 and IL-6, in human mononuclear cells. IL-1 and IL-6 protein expression was significantly enhanced by thrombin (P<.05), as determined by enzyme-linked immunosorbent assay (ELISA). Treating mononuclear cells with the PAR-1 peptide, SFLLRN, has effects similar to those of thrombin. Thus, it appears that these thrombin effects are mediated through activation of PAR-1. These results confirm that thrombin is a strong activator of monocytes and could be involved in angiogenesis by inducing cytokines that could enhance the angiogenic process in tissue repair.

Thrombin peptide TP508 accelerates closure of dermal excisions in animal tissue with surgically induced ischemia.

TP508 is a synthetic peptide corresponding to amino acids 508 through 530 of human prothrombin. We previously demonstrated that a single topical application of TP508 stimulates revascularization and healing of acute incisional and excisional wounds in normal, healthy rat skin. To determine if TP508 would enhance wound healing in ischemic skin, we used bipedicle flaps, cranially based flaps, and free grafts to surgically create ischemic regions on the backs of rats. Full-thickness, circular excisions were made within the flaps or grafts and immediately treated with a single application of saline +/- TP508 (0.1 microg/wound). Compared to wound closure in normal skin, ischemic skin wounds exhibited delayed closure, and the length of delay correlated with the degree of surgically induced ischemia. TP508 significantly accelerated closure in both normal and ischemic skin, resulting in closure rates that were increased within the first 7 days of wounding by 30% in normal tissue and bipedicle flaps, 50% in cranially based flaps, and 225% in free grafts. Moreover, in both flap models, TP508 restored the rate of closure to a rate approximating the control rate observed in normal skin. Histological comparisons of wound tissue from normal skin and cranially based flaps showed that ischemia reduced early recruitment of inflammatory cells at day 1 but increased inflammatory cell numbers in wound beds at day 14. TP508 treatment of ischemic flap wounds significantly increased early inflammatory cell recruitment and restored the normal rapid resolution of the inflammatory phase. In addition, at day 7, TP508-treated wounds appeared to have an increased number of large functional blood vessels compared to saline controls. These studies support the potential efficacy of TP508 in treating ischemic wounds in humans.

Thrombin peptide, TP508, induces differential gene expression in fibroblasts through a nonproteolytic activation pathway.

Prior studies have shown that synthetic peptides representing the domain of thrombin responsible for high-affinity binding to fibroblasts stimulate chemotactic and cell proliferative signals through a nonproteolytic mechanism. One of these peptides, TP508, has recently been shown to be chemotactic for neutrophils, to enhance collagen accumulation in wounds, to enhance revascularization of wounds, and to accelerate the healing of incisional and open wounds in normal animals and in animals with impaired healing. To determine whether TP508 activates the proteolytically activated receptor for thrombin (PAR1), or the signals that are activated by PAR1, we treated human fibroblasts with TP508 and the PAR1-activating peptide, SFLLRNP, and analyzed the effects of these peptides on gene expression using differential display reverse transcriptase polymerase chain reaction. TP508 induces expression of a number of specific message fragments with short tyrosine kinase-like domains that are not induced by SFLLRNP. Sequencing full-length clones prepared by Marathon extension of TP508-induced fragments revealed that among the induced transcripts, there was a sequence with 88% homology to human annexin V. Northern analysis with authentic annexin V cDNA confirms that TP508, but not SFLLRNP, induces expression of annexin V in human fibroblasts. These results demonstrate that TP508 activates a cellular response separate from that activated through PAR1 and supports the hypothesis that TP508 acts through a separate nonproteolytically activated thrombin receptor that may be responsible for high-affinity thrombin binding and for nonproteolytic signals that are required for thrombin stimulation of cell proliferation.

Thrombin receptor expression and responsiveness of human monocytic cells to thrombin is linked to interferon-induced cellular differentiation.

Human thrombin has been shown to stimulate monocyte chemotaxis, phagocytosis, and interleukin (IL8) production, but the mechanisms responsible for stimulation are not well defined. In some cells, thrombin stimulation of proliferation appears to require both cleavage of the proteolytically activated receptor for thrombin (PAR1) and activation of a nonproteolytically activated thrombin receptor (N-PAR), while in others activation of either receptor alone may be sufficient for stimulation. We, therefore, have initiated studies to address thrombin receptor expression and cell responsiveness to thrombin in interferon gamma (IFNgamma)-differentiated and nondifferentiated U937 monocytic cells. Northern blot analysis shows that PAR1 expression is upregulated upon differentiation. Experiments with biotinylated and 125I-thrombin show that specific thrombin binding is dramatically increased by differentiation although it is not clear if this binding is to PAR1 or to a separate binding component such as N-PAR which is present on fibroblasts and other cells. Addition of thrombin at concentrations of 1-10 microg/ml (30-300 nM, concentrations where specific thrombin binding is observed) stimulates proliferation of IFNgamma-differentiated U937 cells but not of undifferentiated U937 cells. Thrombin also stimulates interleukin-6 (IL6) production in IFNgamma-differentiated U937 cells. Moreover, thrombin induces high levels of IL6, interleukin-1beta (IL1beta), and tumor necrosis factor-alpha (TNF alpha) production by peripheral blood mononuclear cells (PBMC) and monocytes. These results show that differentiated U937 cells and mature PBMC are responsive to thrombin whereas nondifferentiated U937 are not. Further, this responsiveness appears to correlate with expression of PAR1 and to a dramatic increase in specific thrombin binding. That thrombin stimulates cytokine production and proliferation in populations of differentiated monocytes suggests that thrombin may be an important regulator of inflammation and wound healing.

Thrombin modulation of natural killer activity in human peripheral lymphocytes.

In addition to its pivotal role in the coagulation cascade, thrombin is mitogenic for fibroblasts and endothelial cells, and activates a number of inflammatory cells including monocytes and T-lymphocytes. To determine if other immune functions are modulated by thrombin and if this modulation is direct or indirect, we investigated whether highly purified human alpha-thrombin affects natural killer (NK) and lymphokine-activated killer (LAK) cell-mediated cytotoxicity. Thrombin enhanced NK cell-mediated cytotoxicity by more than 60% and enhanced IL-2 production and NK 3.3 cell responsiveness to IL-2. Unexpectedly, thrombin and the receptor activating "tethered ligand" domain of the thrombin receptor (TRP-7:SFLLRNP) inhibited LAK cell-mediated cytotoxicity by 50%. DIP-thrombin (a proteolytically inactive form of alpha-thrombin) had no inhibitory activity, suggesting that proteolytic activation of thrombin receptor is requisite for inhibition. These results indicate that cell-mediated cytotoxicity may be enhanced by thrombin through a mechanism involving stimulation of cytokine production and NK cell responsiveness, but that activation of thrombin receptor may also inhibit cytotoxic effects of LAK cells. The role of this dual regulation in processes of cell surveillance, would healing, and inflammation remains to be determined.

Thrombin induces IL-6 production in fibroblasts and epithelial cells. Evidence for the involvement of the seven-transmembrane domain (STD) receptor for alpha-thrombin.

alpha-Thrombin is a multifunctional serine protease that has an important role in the coagulation cascade, wound healing, and inflammatory response. In this study, we show that thrombin induces IL-6 production in human epithelial cells and fibroblasts. ELISA and Northern blot analyses showed that physiologic concentrations of thrombin (0.1-1 micrograms/ml) induced IL-6 production in human lung fibroblasts, skin fibroblasts, and epithelial cells. Hirudin, a thrombin inhibitor, completely blocked IL-6 induction by thrombin. Treatment of fibroblasts with inactivated diisopropylphosphofluoridate (DIP)-alpha-thrombin, gamma-thrombin, or trypsin had no effect on IL-6 production. In contrast, treatment with the thrombin-tethered ligand receptor peptide TRP-7 (SFLLRNP) induced IL-6 production, but at lower levels than that induced by native alpha-thrombin. Finally, IL-6 pretreatment of lung or skin fibroblasts resulted in the enhanced production of IL-6 following exposure to thrombin. These results suggest that fibroblasts and epithelial cells may represent a significant source of IL-6 in the inflammatory response to tissue injury, and that cytokine production is an important biologic consequence of thrombin's interaction with its seven-transmembrane domain (STD) receptor.

Mouse fibroblasts defective in thrombin mitogenesis possess functional proteolytically activated receptor for thrombin: requirement for a second signaling pathway.

Thrombin mitogenesis in fibroblasts requires two distinguishable subsets of signals; one generated by proteolytic cleavage, the other by high-affinity cell surface binding. Characterizing two closely related mouse embryo (ME) cell lines with high numbers of thrombin binding sites, we found that one line, B11-A, responds mitogenically to thrombin, epidermal growth factor (EGF), and serum, whereas the B11-B cell line is responsive to EGF and serum, but not to thrombin. The B11-B defect responsible for loss of thrombin responsiveness is not due to differences in the number of high-affinity binding sites, the affinity of thrombin binding to these sites, or to differences in cell surface expression of proteolytically activated receptors for thrombin (PART). The defect is also not associated with an inability of thrombin to activate PART since thrombin stimulates the cleavage-dependent induction of the proto-oncogene c-fos in both B11-A and B11-B cells. Various combinations of thrombin, synthetic thrombin receptor peptide, TRP-14 (SFFLRNPGENTFEL), platelet-derived growth factor (PDGF), and phorbol 12-myristate 13-acetate (PMA) were used to better define the defect in thrombin-mediated mitogenesis in B11-B cells. Direct activation of protein kinase C with PMA in combination with thrombin did not overcome B11-B nonresponsiveness. However, mitogenic responsiveness was regained in B11-B cells by simultaneous addition of PDGF and either thrombin or TRP-14. Therefore, the B11-B defect may involve a set of signals initiated by nonproteolytic thrombin interactions distinct from those initiated by PART, but related to the downstream signals initiated by the tyrosine kinase-associated growth factors, EGF and PDGF.

Thrombin stimulates fibroblast-mediated collagen lattice contraction by its proteolytically activated receptor.

Fibroblast contraction is proposed to play an important role in tissue contraction during events such as wound healing. Thrombin has been implicated to promote force generation in fibroblasts; however, its extracellular mode of action is unclear. The purpose of this study was to determine the role thrombin and the activation of its receptor plays in promoting the contraction of human fibroblasts in an in vitro collagen lattice contraction assay. Human alpha-thrombin promoted fibroblast contraction in a dose-dependent manner with maximal activity at 0.2 nM. In contrast, both hirudin-alpha-thrombin and D-phenylalanyl-L-propyl-L-arginyl chloromethyl ketone-alpha-thrombin, which lack enzymatic activity, failed to elicit fibroblast contraction. Thus, the enzymatic activity of thrombin appears to be necessary for promotion of fibroblast contraction. Northern analysis confirmed that these human fibroblasts expressed mRNA for the human alpha-thrombin receptor. Moreover, the synthetic peptide (SFLLRNPND-KYEPF) representing the "tethered ligand" portion of the activated alpha-thrombin receptor promoted fibroblast contraction, while a control isomer peptide, in which the first two amino acids were reversed, failed to elicit this response. These findings indicate that alpha-thrombin promotes the contraction of adult human fibroblasts and that cleavage of the human alpha-thrombin receptor is sufficient to produce this response.

Thrombin enhances T cell proliferative responses and cytokine production.

Human alpha-thrombin, in addition to its procoagulant activity, is a mitogen for fibroblasts and endothelial cells and a chemotactic agent for monocytes. To further understand the complex physiological functions of thrombin, we investigated whether thrombin has any immunoregulatory function with regard to T cell activation. Using highly purified human alpha-thrombin and peripheral blood mononuclear cells (PBMC), we investigated whether thrombin has any effect on cytokine production and/or proliferation induced by different T cell stimuli. At physiological concentrations (1-10 micrograms/ml, 30-300 nM), thrombin significantly enhances T cell proliferation in response to mitogens, superantigens, alloantigens, and anti-CD3 stimulation. Enhanced proliferation was associated with increased IL2 and IL6 production and with an increase in the number of IL2r+ (CD25)-bearing T cells. Thrombin alone was not mitogenic nor did it induce IL2 production or increase the number of IL2r+ T cells. However, PBMC exposed to thrombin alone produced high levels of IL6. Thrombin also enhanced IL2-induced proliferation of murine and human IL2-dependent cell lines. These results suggest that thrombin may play an important role in regulating cell-mediated immunity.

Acceleration of soft tissue repair by a thrombin-derived oligopeptide.

Augmentation of thrombin-modulated chemotaxis and mitogenic activity within the early phase of soft tissue repair is now possible. Identification of high-affinity thrombin receptor binding domains within thrombin has enabled the synthesis of a family of peptides which interact with thrombin receptors and enhance in vitro mitogenesis. A single (5.0 micrograms/wound) application of the thrombin receptor-activating peptide (P517-30) significantly increased wound breaking strength from Day 5 (31% over controls) to Day 12. Two models of impaired healing created by radiotherapy (RT) were used to elucidate possible mechanisms of P517-30 action. Although P517-30 did not completely overcome the RT-induced healing impairments, it increased breaking strength under conditions of penetrating whole body RT-induced pancytopenia by 22% and of nonpenetrating surface RT-induced dermal cell damage by 42%. This suggests that P517-30 directly stimulates resident endothelial cells, fibroblasts, or other cells to overcome dermal and circulating monocytic deficits. These results suggest a method to accelerate wound healing with potential clinical applications and emphasize the activity of thrombin as a growth factor.

Enhancement of incisional wound healing and neovascularization in normal rats by thrombin and synthetic thrombin receptor-activating peptides.

To better define thrombin-receptor interactions, we synthesized human thrombin peptides and identified binding-domain peptides that bind thrombin receptors and activate mitogenic signals (Glenn, K.C., G.H. Frost, J.S. Bergmann, and D.H. Carney. 1988. Pept. Res. 1:65-73). Treatment of full dermal dorsal incisions with a single topical application of thrombin receptor-activating peptide (TRAP-508) or human alpha-thrombin in saline enhances 7-d incisional breaking strength in normal rats up to 82% or 55% over saline-treated controls, respectively. Control wounds require approximately 11.5 d to achieve breaking strength equivalent to TRAP-treated wounds at day 7. Thus, a single application of TRAP accelerates healing, shifting the time course forward by up to 4.5 d. Histological comparisons at day 7 show more type I collagen, less evidence of prolonged inflammation, and an increase in number and maturity of capillaries in TRAP- and thrombin-treated incisions. Angiograms also show 50-65% more functional vascularization going across thrombin- and TRAP-treated surgical incisions. Thus, alpha-thrombin and thrombin peptides, such as those released following injury, appear to initiate or enhance signals required for neovascularization and wound healing. The ability to accelerate normal wound healing events with synthetic peptides representing receptor binding domains of human thrombin may offer new options for management of wound healing in man.

Thrombin, epidermal growth factor, and phorbol myristate acetate stimulate tubulin polymerization in quiescent cells: a potential link to mitogenesis.

Previous studies suggest that alterations in the microtubule (MT)-tubulin equilibrium during G0/G1 affect mitogenesis. To determine the effect of growth factors on the MT-tubulin equilibrium, we developed a radioactive monoclonal antibody binding assay (Ball et al.: J. Cell. Biol. 103:1033-1041, 1986). With this assay, 3H-Ab 1-1.1 binding to cytoskeletons in confluent populations of cultured cells is proportional to the number of tubulin subunits polymerized into MTs. We now show that purified alpha-thrombin increases 3H-Ab 1-1.1 binding to cytoskeletons of serum-arrested mouse embryo (ME) fibroblasts from 1.5- to 3-fold. This stimulation is dose-dependent and correlates with concentrations of thrombin required for initiation of DNA synthesis. Other mitogenic factors, epidermal growth factor (EGF) and phorbol 12-myristate 13-acetate (PMA), also stimulate MT polymerization. Addition of colchicine (0.3 microM) eight hours after growth factor addition, blocks stimulation of 3H-thymidine incorporation by thrombin, EGF, or PMA, suggesting that tubulin polymerization or subsequent events triggered by MT polymerization are required for cells to enter a proliferative cycle. Consistent with models for autoregulation of tubulin synthesis, thrombin, EGF, and PMA all increase tubulin synthesis 9 to 15 hr after growth factor addition, raising the possibility that the decrease in free tubulin and subsequent stimulation of tubulin synthesis is linked to progression of cells into a proliferative cycle. Colchicine addition to these cells also stimulates DNA synthesis, but colchicine-stimulated cells enter S phase 6 to 8 hr later than those stimulated by growth factors. This delayed stimulation may be related to the time required for degradation of tubulin-colchicine complexes below a critical level. These data suggest that regulation of cell proliferation may be linked to increased MT polymerization and the resulting decrease in free tubulin pools.

Organ-derived microvessel endothelial cells exhibit differential responsiveness to thrombin and other growth factors.

To investigate the relationship between endothelial cells and organ-associated vascular physiology, microvascular endothelial cells were isolated from murine brain, lung, and liver tissues. During culture, these endothelial cells maintained certain differentiated characteristics common to all endothelial cells, but also showed organ-specific characteristics, with distinct patterns of responsiveness to various growth factors. Microvascular endothelial cells from all organs responded to endothelial cell growth factor (ECGF), but lung (LE-1) and brain (MBE-12) endothelial cells showed different responsiveness to thrombin (10-60 nM), combinations of thrombin and ECGF, or thrombin and extracellular matrix. Liver sinusoidal endothelial cells (HSE) were relatively unresponsive to thrombin, but were the most responsive of the endothelial cells to EGF. Endothelial cells isolated from lung and brain, where fluxes in vascular permeability are observed following injury, showed dramatic morphological alterations in response to nanomolar concentrations of thrombin. These cells also exhibited the highest amount of 125I-thrombin binding at these concentrations. Scatchard analysis of 125I-thrombin binding indicated that LE cells have the highest affinity for thrombin, followed by MBE, with HSE exhibiting significantly lower affinity. The binding of 125I-thrombin to these cells was inhibited by the TR-9 monoclonal antibody directed against fibroblast high-affinity thrombin receptors involved in thrombin-stimulated mitogenesis. The results suggest that the differences in growth stimulation observed between organ-derived endothelial cells in response to thrombin, ECGF, and EGF may relate to differential expression of receptors for these factors. These observations demonstrate yet another aspect of the functional heterogeneity of the microvascular endothelium.