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.

Cell-specific heterogeneity in sensitivity of phosphatidylinositol-anchored membrane antigens to release by phospholipase C.

Cell surface antigens thought to be linked to the membrane via phosphatidylinositol (PI) are incompletely, and variably, released by treatment with phosphatidylinositol-specific phospholipase C (PI-PLC). The basis for this was investigated with cloned tumor cell lines and PI-PLCs isolated from two species of bacteria. Residual Thy-1 antigen, which was detectable by flow cytometry, remained on all thymoma cell lines after exposure to very high concentrations of either purified enzyme. A majority of the presumptive PI anchored molecules on all of the cell lines was sensitive to release by PI-PLC derived from Bacillus thuringiensis. However, cell lines differed dramatically in the ease with which PI-PLC from Staphylococcus aureus liberated the same surface antigens. This heterogeneity was determined at the single cell level because at least five different PI-anchored antigens exhibited similar behavior on a given cell line or transfected subclones of it. The two phospholipases differed with respect to molecular mass, serological cross-reactivity and sensitivity to inhibition by NaCl and detergents. These observations suggest that the two types of PI-PLC may have distinct substrate specificities or sensitivities to environmental conditions which account for the difference in their ability to act on PI-anchored proteins in particular cell types. Such enzymes should continue to be important tools for investigating the method and significance of attachment of lymphocyte surface glycoproteins. In particular, the S. aureus PI-PLC can be used to demonstrate and investigate a previously unrecognized heterogeneity in cells which express PI-anchored molecules.

Removal of lymphocyte surface molecules with phosphatidylinositol-specific phospholipase C: effects on mitogen responses and evidence that ThB and certain Qa antigens are membrane-anchored via phosphatidylinositol.

We reported previously that the Thy-1 antigen was released from murine thymocytes and thymoma cells by S. aureus-derived phosphatidylinositol-specific phospholipase C (PI-PLC). It is therefore part of a small group of proteins known to use a unique form of membrane attachment. This finding has now been extended in studies with peripheral lymphocytes and additional leukocyte markers. Retention of viability and responsiveness to LPS were excellent in PI-PLC-treated spleen cells and there was no appreciable effect on lectin-binding surface glycoproteins. Thy-1 regeneration was insignificant on unstimulated spleen cells within 24 hr of treatment, but nearly complete at this time with a continuously dividing cell line. In contrast to the result with LPS, responses to the mitogens Con A, PHA, and PWM were virtually eliminated. Of more than 40 monoclonal antibodies tested, only staining with ThB and particular Qa specificities were diminished by PI-PLC treatment. The latter included Qa-2, Qa-4, Qa-5, and possibly also Qa-6, whereas Qa-1, TLa, and other class I and class II histocompatibility antigens were unaffected. Although the validity of the Qa results seems assured by the total PI-PLC resistance of many other lymphocyte antigens, the pattern of release was notably different from that observed with Thy-1 and ThB. That is, the density of Qa-2 was usually unchanged on a subpopulation of Qa-2-positive cells. This raises interesting questions about lymphocyte heterogeneity and flexibility in the use of this form of surface protein anchoring. Glycosyl-phosphatidylinositol-linked proteins may be functionally significant in immunological responses, and this experimental approach should continue to be valuable for their identification and characterization.

Initiation of DNA synthesis by human thrombin: relationships between receptor binding, enzymic activity, and stimulation of 86Rb+ influx.

Stimulation of amiloride-sensitive sodium (Na+) influx and the subsequent activation of NA+, K+-ATPase by serum or growth factors have been implicated as early events leading to initiation of cell proliferation. We recently demonstrated that amiloride inhibits thrombin-initiated DNA synthesis not by inhibiting an early event occurring during the first 8 hr, but rather by inhibiting some later event 8 to 12 hr after thrombin addition. To further probe the relationship between stimulation of ion influx and initiation of cell proliferation, human alpha-thrombin was converted to gamma-thrombin, nitro-alpha-thrombin, and diisopropylphospho (DIP)-alpha-thrombin. These derivatives retain either the capacity to bind cell surface alpha-thrombin receptors or thrombin esterase activity, but they do not initiate DNA synthesis. At low concentrations of alpha-thrombin or the various thrombin derivatives, only alpha-thrombin stimulates 86Rb+ influx, suggesting a correlation between stimulation of influx and the ability of these derivatives to initiate DNA synthesis. Concentrations of a DIP-alpha-thrombin that saturate the alpha-thrombin receptors (up to 2 micrograms/ml) do not stimulate either the early or late influx of 86Rb+, indicating that DIP-alpha-thrombin binding alone is not sufficient to stimulate ion fluxes. High concentrations of either gamma-thrombin or nitro-alpha-thrombin, however, stimulate both early and late 86RB+ uptake but do not initiate DNA synthesis. These results demonstrate that events leading to both the early and late stimulation of 86Rb+ influx by themselves are not sufficient to initiate cell proliferation. Thus, initiation may require a combination of events that can be independently regulated by different transmembrane signals.

Initiation of proliferative events by human alpha-thrombin requires both receptor binding and enzymic activity.

To determine the role of thrombin high-affinity receptor occupancy and enzymic activity in thrombin initiation of cell proliferation, we have utilized thrombin derivatives which separate these functions. We previously showed that enzymically active gamma-thrombin stimulates ion fluxes without binding to high-affinity sites, whereas proteolytically inhibited DIP-alpha-thrombin which binds to high-affinity receptors does not. Since neither derivative initiates DNA synthesis by itself, this suggested that two separate sequences of events might be necessary for a complete initiation signal. We now report that the combination of DIP-alpha-thrombin and gamma-thrombin initiate DNA synthesis and cell proliferation to levels approaching the maximal initiation by native alpha-thrombin. This combinatory effect is dose-dependent for both gamma-thrombin and DIP-alpha-thrombin in the same concentration range as alpha-thrombin alone. Thus, these same concentrations of alpha-thrombin alone may be required to initiate each sequence of events. The combinatory stimulation could be achieved even if the derivatives were added individually up to 8 hr apart. Moreover, preincubation with either derivative shortened the lag period for initiation of DNA synthesis by native alpha-thrombin. These results indicate that both receptor occupancy and enzymic activity are necessary for thrombin initiation of cell proliferation and that each action initiates a sequence of early events which moves the cell forward toward entry into a proliferative cycle.

Demonstration of a late amiloride-sensitive event as a necessary step in initiation of DNA synthesis by thrombin.

Amiloride, a Na+ influx inhibitor, has been shown to inhibit initiation of DNA synthesis by thrombin in mouse embryo fibroblast-like cells. Long exposures (24 hr) to high concentrations of amiloride inhibited incorporation of thymidine into the DNA of both thrombin-stimulated and nonstimulated cells, suggesting that this inhibition might not be specific for thrombin-initiated DNA synthesis. Fluorescence microscopy and spectrofluorimetry showed that amiloride was internalized with an apparent mitochondrial association and that the internalized amiloride was readily released from the cells after removing amiloride from the medium. Based on this reversibility, cells were exposed to amiloride for short periods of time during thrombin treatment to determine the temporal relationship between any amiloride-sensitive event(s) and initiation of DNA synthesis. The presence of amiloride (100 microM) during a 12-hr exposure to thrombin did not block thrombin-initiated DNA synthesis or cell division but did delay the onset of DNA synthesis and the peak of thymidine incorporation into DNA by approximately 3 hr, suggesting that early initiation events might proceed in the presence of amiloride. 86Rb+ transport studies demonstrated that in this system ouabain-sensitive K+ uptake via the Na, K-ATPase was stimulated by thrombin during both an early and a late period. This stimulation was amiloride-sensitive under the same conditions used for growth experiments, suggesting that amiloride was inhibiting thrombin-stimulated Na+ transport in this system. Additional experiments showed that exposing cells to amiloride only during the first 8 hr after thrombin addition did not inhibit initiation. The presence of amiloride from 8-12 hr after thrombin addition maximally inhibited thrombin-stimulated DNA synthesis. Together these results demonstrate that amiloride inhibits thrombin-initiated DNA synthesis not by inhibiting an early event occurring during the first 8 hr, but rather by inhibiting some later event 8-12 hr after thrombin addition.