The non‐proteolytically active thrombin peptide TP508 stimulates angiogenic sprouting

Abstract

Thrombin is a serine protease that promotes platelet aggregation, blood coagulation, and tissue repair. A peptide derived from a non-proteolytically active region of thrombin, TP508, also promotes tissue repair and increased vascularity, yet does not activate platelet and inflammatory cascades. TP508 binds to cells with high affinity and stimulates cells independent of the proteolytically active thrombin receptors (PARs) and thus is considered to activate a non-proteolytically active receptor (non-PAR) pathway. Using a model of angiogenic sprouting, we further defined the angiogenic potential of TP508 and investigated the role of non-proteolytic, thrombin-mediated pathways in angiogenesis. The assay involves measuring angiogenic sprouting from cultured, intact microvessel fragments. In this assay, TP508 stimulated angiogenic sprouting to an extent similar to or greater than the potent angiogenic factor, VEGF. However, TP508 had no significant effect on the number of sprouts that formed per vessel. In contrast to TP508, proteolytically active receptor agonists had no effect or inhibited angiogenic sprouting. The increased sprouting activity stimulated by TP508 was VEGF dependent but did not involve an increase in VEGF mRNA expression above baseline levels. These results suggest that TP508 acts early in angiogenesis and directly on microvascular cells to accelerate sprouting, but not to induce more sprouting, in a manner different than the intact thrombin molecule.

Satellite cell-mediated angiogenesis in vitro coincides with a functional hypoxia-inducible factor pathway

“…Angiogenic growth of MVF in collagen gels is characterized by the formation of smooth vascular sprouts that are morphologically distinct from the rough, smooth muscle-associated appearance of the parent vessel (15,28,38). The sprouting assay has been described previously (38).…”

“…MVF were isolated from male SpragueDawley rats as previously described (28,38), with slight modifications. Briefly, after rats were killed, epididymal fat pads were collected, minced, and digested in a collagenase solution (2 mg/ml in PBS containing 0.1% BSA) for 8 -10 min.…”

“…The sprouting assay has been described previously (38). Briefly, images of phase contrast microscopic fields (ϫ10 or ϫ20) of selected fragments from each gel were captured using a digital camera.…”

Thrombin Peptide TP508 Stimulates Rapid Nitric Oxide Production in Human Endothelial Cells

“…TP508 has previously been shown to stimulate tissue revascularization following injury [7,8,10,12,20] and angiogenic sprouting in ex vivo cultures of microvessel fragments [21]. Recent in vivo and long-term culture experiments showed that TP508 reverses endothelial dysfunction and increases NO production in endothelial cells [33].…”

“…Angiogenesis, the process of new blood vessel formation from preexisting ones, is comprised of several discrete steps including dissociation of matrix, endothelial cell migration, proliferation and tube differentiation [19]. TP508 stimulated early revascularization in dermal and bone injury models [7,8,10,12,13,20], and stimulated vascular sprouting in ex vivo cultures of microvessel fragments [21]. Thus, TP508 effects on tissue repair appear to be linked to its stimulation of angiogenesis.…”