WO2009002811A2

WO2009002811A2 - Therapeutic platelet compositions and methods

Info

Publication number: WO2009002811A2
Application number: PCT/US2008/067592
Authority: WO
Inventors: Dennis Orgill; Giorgio Pietramaggiori; Giannoula Klement; David Cervi; Judah Folkman
Original assignee: Brigham and Womens Hospital Inc; Boston Childrens Hospital
Current assignee: Brigham and Womens Hospital Inc; Boston Childrens Hospital
Priority date: 2007-06-22
Filing date: 2008-06-20
Publication date: 2008-12-31
Anticipated expiration: 2009-12-22
Also published as: WO2009002811A3

Abstract

Provided are methods and compositions for the therapeutic delivery of angiogenesis-regulating factors to in vivo sites in need thereof. The methods and compositions relate generally to the use of platelets bearing one or more exogenous angiogenesis regulating factors. In one aspect, described are methods of delivering an angiogenesis-regulating factor, the method comprising contacting a tissue with a composition comprising isolated platelets comprising an exogenous angiogenesis regulating factor. The methods can be employed for example, to enhance wound healing, or alternatively, to inhibit tumor growth. In another aspect, therapeutic platelet compositions and kits are provided in which the platelets bear one or more exogenous angiogenesis regulating factors. Also provided are methods of modulating the release angiogenesis promoting and inhibiting factors by platelets using agonists and/or antagonists of platelet protease-activated receptors PAR-1 and PAR-4.

Description

THERAPEUTIC PLATELET COMPOSITIONS AND METHODS

[0001] This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application Serial No. 60/936,921, filed June 22, 2007, the entirety of which is incorporated herein by reference.

BACKGROUND

[0002] Early in wound healing, angiogenesis is essential to initiate and support the repair process. New, small blood vessels form in the granulation tissue, and are a hallmark of tissue healing. Once healing has ceased, endothelial cells in the wound bed revert to a quiescent phenotype, demonstrating that timely switching between pro- and anti-angiogenesis is necessary to initiate and terminate the healing process, respectively¹.

[0003] Wounds and tumor lesions share many biological mechanisms with respect to their vascular component. A tumor can be viewed as "a wound that never heals", a parallel concept suggested by Harold Dvorak in 1986.² Angiogenesis is the crucial component to compel a wound to heal and a tumor to grow.³' ⁴ In cancer, the critical balance between pro- and anti- angiogenic factors has been postulated to keep a tumor in a state of dormancy, and once this balance is skewed in favor of pro-angiogenic factors, it has been postulated that tumor expansion ensues as the vascular supply concomitantly increases. Platelets orchestrate the phases of healing with several pro- and anti- angiogenic growth factors, exposed at the appropriate time. The platelet proteome has recently been shown to change accordingly to the environment, such as in the presence of tumors in vivo. These changes have since been suggested to partake in tumor growth and metastasis.

[0004] Circulating blood components, such as platelets and leukocytes interact with activated endothelium and exposed collagen matrix; environments present in any typical wound. It is in this conducive environment that platelets are found in high concentrations to orchestrate wound healing.^5"8 Recently, a biochemical 'cross-talk' between platelets and tumor cells has been described whereby cancer cells have the capacity to alter the levels of platelet consituitents,⁵ primarily angiogenenic factors. Overall, tumor angiogenesis and meta formation may be governed, in part, by the presentation of factors by platelets. 5, 10 [0005] Several angiogenesis regulating proteins carried by platelets are present at different times during wound healing, and have been shown to be stored in separate (pro- and antiangiogenic) alpha-granule compartments in the platelets cytoplasm. Evidence indicates that a protease activated receptors facilitates a selective release of pro- or anti-angiogenic factors from the platelets. The separate release of inhibitors and stimulators of angiogenesis is thought to a timely and orchestrated release of individual proteins needed for angiogenesis, rather than a single bolus delivery of all proteins contained in platelets as would be consistent with platelet degranulation. Clotted or frozen platelets do not have this same effect. In addition, platelet levels of growth factors and cytokines are also variable^46"49. The concentration of some angiogenic growth factors, such as VEGF, bFGF, PDGF and PF-4, in platelets (but not in plasma) changes as a function of the stage of tumor growth⁴⁶' ^{48> 49}.

[0006] Under normal conditions, platelets store more than 14 angiogenic growth factors, including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), angiopoietin-1 (ANG-I), insulin-like growth factor- 1 (IGF- 1), epidermal growth factor (EGF) and platelet derived growth factor (PDGF).¹¹ VEGF is one of the most studied angiogenic factors. Depending on the particular isoform present in the blood stream, which governs the degree of heparin-binding affinity, VEGF may be presented or concentrated at the tumor site via platelets.¹² Platelets also harbor inhibitors of angiogenesis, including platelet factor-4 (PF-4), thrombospondin-1 (TSP-I), transforming growth factor beta-1 (TGF-betal), plasminogen activator inhibitor type-1 (tPA), alpha₂- antiplasmin and alpha₂-macroglobulin. All have been shown to participate in wound healing in healing-impaired animal models and in human, chronic wounds.¹⁴' ^{13> 14} Thus, the use of platelet concentrates as a treatment option of recalcitrant wounds has become common practice in surgery.¹⁵' ¹⁶ Because they avoid potential infection and rejection problems involved with human blood products, a preferred form of platelet concentrate includes autologous platelet gels (APG), or platelet preparations prepared from the patient's own blood (see, e.g., Horn et al., 2007, Arch. Facial Plast. Surg. 9: 174-183).

[0007] Pro- and anti-angiogenic growth factors and cytokines are required to modulate vascular growth, but they may not be sufficient for sustaining angiogenesis. In in vivo experimental models in which platelets were made dysfunctional growing blood vessels exhibited moderate maturation and functionality. Similarly, previous work corroborates this finding, because the use of recombinant growth factors or sonicated platelets on experimental full-thickness diabetic wounds did not match up with the pro-angiogenic properties of intact platelets. Platelet open canallicular system and the integrity of the membranes may be the gate controlling uptake and delivery of the various proteins that regulate angiogenesis.

[0008] Ma et al. describe the involvement of protease activated receptors PAR-I and PAR-4 in the counter-regulation of the release of VEGF and endostatin from human platelets (Ma et al., 2005, Proc. Natl. Acad. Sci. U.S.A. 102: 216-220). Specifically, Ma et al. teach that human platelets contain endostatin, and that its release can be triggered by activation of PAR- 4 but not PAR-I. Ma et al. also teach that PAR-I activation leads to the suppression of endostatin release but also to stimulation of the release of the proangiogenic factor VEGF, while PAR-4 activation stimulates endostatin release and suppresses release of VEGF. Ma et al. conclude that PAR-I and PAR-4 appear to act in a counter-regulatory manner to modulate release of factors regulating angiogenesis.

SUMMARY OF THE INVENTION

[0009] There is a great need for methods and compositions that enhance wound healing. Such methods and compositions find use in a range of applications, including, for example, promoting the rapid healing of surgical wounds, including, but not limited to plastic surgery, as well as promoting the healing of chronic wounds, e.g., decubitus ulcers and diabetic ulcers, among others. The methods and compositions described herein relate in part to the discovery that platelets exposed to different environments or factors before application to a wound surface can dramatically influence the healing of the wound. For example, platelets exposed to a tumor environment have been found to enhance the rate of wound healing in animals without tumors. This promotion of wound healing is mediated at least in part by the delivery of angiogenesis regulating factors by the platelets.

[0010] It is recognized herein that platelets can be used to deliver exogenously added angiogenesis regulating factors directly to a wound site to promote healing at that site. Thus, described herein is a composition for promoting wound healing, the composition comprising platelets comprising an exogenous angiogenesis promoting factor and a pharmaceutically acceptable carrier.

[0011] In one embodiment of this aspect and all other aspects described herein, the platelets are autologous to the individual. [0012] In another embodiment of this aspect and all other aspects described herein, the angiogenesis regulating factor is selected, for example, from the group consisting of agents that activate the VEGF pathway, agents that activate the neuropilin 1 & 2 pathways, VEGF-A and C, bFGF, HGF, angiopoietin-1, insulin-like growth factor- 1, epidermal growth factor, platelet derived growth factor, platelet factor 4, thrombospondin-1, TGF-beta-1, plasminogen activator inhibitor type-1 (PAI-I, alpha2-antiplasmin and alpha2-macroglobulin VEGFRl (flt- 1), VEGFR2 (flk-2), VEGFR3 (flt-4), heparin sulfate proteoglycan, VEGF121, VEGF145, VEGF165, VEGF168, VEGF189, VEGF -B and -D, PLGF 1, PLGF2, HIV-I TAT, Sema-E, Sema-III, Sema-IV, bFGF, PDGFR, EGFR, and IGFR.

[0013] In another embodiment of this aspect and all other aspects described herein, the wound is selected, for example, from a burn, an incision, a laceration, an abrasion, an ulcer, a diabetic wound, a venous stasis wound, a vascular wound, a radiation wound, a steroid wound or a bony defect.

[0014] In another embodiment of this aspect and all other aspects described herein, the wound is a diabetic ulcer or a decubitus ulcer.

[0015] In another embodiment of this aspect and all other aspects described herein, the method further comprises the step of loading the platelets with the exogenous angiogenesis regulating factor. In another embodiment, the loading comprises contacting platelets with the factor.

[0016] In another embodiment of this aspect and all other aspects described herein, the platelets comprise at least two angiogenesis promoting factors.

[0017] In another embodiment, the composition comprises a platelet gel comprising the platelets and the angiogenesis regulating factor. Where this approach is used, the gel can be formed by contacting the platelets with calcium and a platelet activating agent. The platelet activating agent can be selected, for example, from the group consisting of thrombin, collagen, serotonin, ADP, acetylcholine and combinations thereof.

[0018] In another embodiment of this aspect and all other aspects described herein, the angiogenesis promoting factor comprises a PAR-I agonist. The PAR-I agonist can be selected, for example, from the group of TFLLR-NH₂; TFLLRNPNDK-NH₂; SFLLRNPNDKYEPF-NH₂; and SFLLRN-NH₂ [0019] In another embodiment of this aspect and all other aspects described herein, the angiogenesis promoting factor comprises a PAR-4 antagonist. The PAR-4 antagonist can be selected, for example, from the group of transcinnamoyl- YPGKF-NH₂ (tcY-NH₂; Ma et al.) and YD-3, a non-peptide PAR4 antagonist nonpeptide PAR-4 antagonist, YD-3 (ethyl 4-(l- benzyl-lH-indazol-3-yl)benzoate) (2006, Eur. J. Pharmacol. 546: 142-147).

[0020] In another embodiment of this aspect and all other aspects described herein, the platelets further comprise an exogenous angiogenesis inhibiting factor.

[0021] Also described herein is the use of a composition as described herein comprising platelets comprising an exogenous angiogenesis regulating factor and a pharmaceutically acceptable carrier for promotion of wound healing.

[0022] In one embodiment of this aspect, the platelets are autologous to the individual.

[0023] In another embodiment, the angiogenesis regulating factor is selected, for example, from the group consisting of agents that activate the VEGF pathway, agents that activate the neuropilin 1 & 2 pathways, VEGF-A and C, bFGF, HGF, angiopoietin-1, insulin-like growth factor- 1, epidermal growth factor, platelet derived growth factor, platelet factor 4, thrombospondin-1, TGF-beta-1, plasminogen activator inhibitor type-1 (PAI-I, alpha2- antiplasmin and alpha2-macroglobulinVEGFRl (flt-1), VEGFR2 (flk-2), VEGFR3 (flt-4), heparin sulfate proteoglycan, VEGF121, VEGF145, VEGF165, VEGF168, VEGF189, VEGF -B and -D, PLGF 1, PLGF2, HIV-I TAT, Sema-E, Sema-III, Sema-IV, bFGF, PDGFR, EGFR, and IGFR.

[0024] In another embodiment, the wound is selected, for example, from a burn, an incision, a laceration, an abrasion, an ulcer, a diabetic wound, a venous stasis wound, a vascular wound, a radiation wound, a steroid wound or a bony defect.

[0025] In another embodiment the wound is a diabetic ulcer. In an alternative embodiment, the wound is a decubitus ulcer.

[0026] In another embodiment, the platelets are loaded with an exogenous angiogenesis regulating factor.

[0027] In another embodiment, loading comprises contacting platelets with a factor. [0028] In another embodiment of this aspect, the platelets comprise at least two angiogenesis promoting factors.

[0029] In another embodiment, the composition comprises a platelet gel comprising platelets and an angiogenesis regulating factor.

[0030] In another embodiment, the gel is formed by contacting the platelets with calcium and a platelet activating agent.

[0031] In another embodiment, the platelet activating agent is selected, for example, from the group consisting of thrombin, collagen, serotonin, ADP, acetylcholine and combinations thereof.

[0032] In another embodiment of this aspect, the angiogenesis regulating factor comprises a PAR-I agonist.

[0033] In another embodiment, the PAR-I agonist is selected, for example, from the group consisting Of TFLLR-NH₂ , TFLLRNPND K -N H₂, SFLLRNPNDKYEPF-NH₂, and SFLLRN- NH₂.

[0034] In another embodiment of this aspect, the angiogenesis promoting factor comprises a PAR-4 antagonist.

[0035] In another embodiment, the PAR-4 antagonist is selected, for example, from the group consisting of transcinnamoyl- YPGKF-NH₂, and ethyl 4-(l -benzyl- lH-indazol-3- yl)benzoate.

[0036] In another embodiment, the platelets further comprise an exogenous angiogenesis inhibiting factor.

[0037] Further encompassed is the use of a platelet composition comprising an exogenous angiogenesis regulating factor in the preparation of a medicament for the promotion of wound healing.

[0038] In one embodiment of this aspect, the wound is selected, for example, from a burn, an incision, a laceration, an abrasion, an ulcer, a diabetic wound, a venous stasis wound, a vascular wound, a radiation wound, a steroid wound or a bony defect. [0039] In another embodiment, the wound is a diabetic ulcer. In an alternate embodiment, the wound is a decubitus ulcer.

[0040] In another embodiment, the platelets are loaded with an exogenous angiogenesis regulating factor.

[0041] In another embodiment, loading comprises contacting platelets with a factor.

[0042] In another embodiment, the platelets comprise at least two angiogenesis promoting factors.

[0043] In another embodiment, the composition comprises a platelet gel comprising platelets and an angiogenesis regulating factor.

[0044] In another embodiment, the gel is formed by contacting platelets with calcium and a platelet activating agent.

[0045] In another embodiment, the platelet activating agent is selected, for example, from the group consisting of thrombin, collagen, serotonin, ADP, acetylcholine and combinations thereof.

[0046] In another embodiment, the angiogenesis regulating factor comprises a PAR-I agonist.

[0047] In another embodiment, the PAR-I agonist is selected, for example, from the group consisting Of TFLLR-NH₂ , TFLLRNPNDK-NH.., SFLLRNPNDKYEPF-NH₂, and SFLLRN- NH₂.

[0048] In another embodiment, the angiogenesis promoting factor comprises a PAR-4 antagonist.

[0049] In another embodiment, the PAR-4 antagonist is selected, for example, from the group consisting of transcinnamoyl- YPGKF-NH₂, and ethyl 4-(l -benzyl- lH-indazol-3-yl) benzoate.

[0050] In another embodiment, the platelets further comprise an exogenous angiogenesis inhibiting factor.

[0051] Another aspect described herein relates to a method of promoting wound healing in an individual in need thereof, the method comprising the step of contacting a wound with a composition as described herein comprising isolated platelets comprising an exogenous angiogenesis regulating factor, wherein the contacting promotes healing of the wound.

[0052] Also encompassed is a method of delivering angiogenesis regulators to a wound site, the method comprising contacting the wound site with a composition comprising isolated platelets comprising an exogenous angiogenesis regulating factor, whereby the angiogenesis regulating factor is delivered to the wound site.

[0053] Further encompassed is a method of preparing a wound healing composition, the method comprising contacting platelets with an exogenous angiogenesis regulating factor.

[0054] In one embodiment of this aspect and all other aspects described herein, the angiogenesis regulating factor is selected, for example, from the group consisting of agents that activate the VEGF pathway, agents that activate the neuropilin 1 & 2 pathways, VEGF-A and C, bFGF, HGF, angiopoietin-1, insulin-like growth factor- 1, epidermal growth factor, platelet derived growth factor, platelet factor 4, thrombospondin-1, TGF-beta-1, plasminogen activator inhibitor type-1 (PAI-I, alpha2-antiplasmin and alpha2-macroglobulin VEGFRl (flt- 1), VEGFR2 (flk-2), VEGFR3 (flt-4), heparin sulfate proteoglycan, VEGF121, VEGF145, VEGF165, VEGF168, VEGF189, VEGF -B and -D, PLGF 1, PLGF2, HIV-I TAT, Sema-E, Sema-III, Sema-IV, bFGF, PDGFR, EGFR, and IGFR.

[0055] In another embodiment of this aspect and all other aspects described herein, the method further comprises contacting the platelets that have been contacted with an angiogenesis regulating factor with a platelet activating agent. The platelet activating agent can be selected, for example, from the group of thrombin, collagen, serotonin, ADP, acetylcholine and combinations thereof.

[0056] Also encompassed is a kit for the preparation of a wound healing composition, the kit comprising an angiogenesis regulating factor and a platelet preparation.

[0057] Also encompassed is a kit for the preparation of a wound healing composition, the kit comprising an angiogenesis regulating factor and a container for holding and/or incubating platelets. Kits as described herein can further comprise packaging materials therefor and, optionally, instructions for the preparation and/or use of a platelet wound healing composition. Definitions:

[0058] As used herein, the term "angiogenesis" refers to any alteration of an existing vascular bed or the formation of new vasculature which benefits tissue perfusion. This includes the formation of new vessels by sprouting of endothelial cells from existing blood vessels or the remodeling of existing vessels to alter size, maturity, direction or flow properties to improve blood perfusion of tissues.

[0059] As used herein, the term "angiogenesis regulating factor" refers to an agent that modulates angiogenesis. An "angiogenesis regulating factor" regulates the angiogenic process, including but not limited to the following phases of the process: the degradation of the extracellular matrix; cell proliferation; cell migration and structural organization (see, e.g., Kumar et al, 1998, Int. J. Oncology 12:749-757; Bussolino et al., 1997, Trends in Biochem, 22:251-256). Such a factor thus influences the rate or progress of angiogenesis, e.g., initiating angiogenesis, accelerating angiogenesis, or inhibiting angiogenesis. At a minimum, an "angiogenesis regulating factor" positively or negatively influences angiogenesis in a chick chorioallantoic membrane ("CAM") assay performed, for example, as described by Iruela-Arispe et al., 1999, Circulation 100: 1423-1431, which is incorporated herein by reference. By "positively or negatively influences" is meant an at least 10% difference in angiogenesis relative to the absence of that factor. Further detail of this assay is provided herein below.

[0060] The term "angiogenesis regulating factor" encompasses both those factors that occur naturally in vivo and participate in the angiogenic regulatory pathways in vivo, as well as factors or compounds, either derived from naturally-occurring polypeptides or molecules (including, but not limited to fragments of naturally occurring polypeptides, e.g., anti- angiogenic fragments of collagen polypeptides) or otherwise generated or identified, that have angiogenesis-regulating activities.

[0061] In one aspect, an angiogenesis regulating factor is an angiogenesis-promoting factor (also referred to as a proangiogenesis factor). An angiogenesis-promoting factor is one that causes an increase in angiogenic activity, e.g., at least a 10% increase in angiogenic activity as measured by the CAM assay as described by Iruela-Arispe et al., 1999, Circulation 100: 1423-1431, relative to the angiogenic activity observed in the absence of that factor. [0062] In another aspect, an angiogenesis regulating factor is an angiogenesis-inhibiting factor (also referred to as an antiangiogenesis factor). An angiogenesis-inhibiting factor is one that causes a decrease in angiogenic activity, e.g., at least a 10% decrease in angiogenic activity as measured by the CAM assay as described by Iruela-Arispe et al., 1999, Circulation 100: 1423-1431, relative to the angiogenic activity observed in the absence of that factor.

[0063] As used herein, the term "promotes healing" means that treatment with a given agent decreases the time required for 90% wound closure of a full-thickness skin wound by at least 1 day relative to a control wound not treated with that agent. Preferably, a treatment that promotes healing will decrease the time necessary to heal a full thickness wound by 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, or even 7 days (one week) or more.

[0064] An alternative measure of wound healing is measurement of cell proliferation at a given time after treatment (see the Examples herein below). Such measurement generally requires a biopsy (i.e., another wound), however, which is clearly not preferred for the evaluation of treatment efficacy in human subjects outside of, for example, a clinical trial setting. Thus, measurements based on cell proliferation at the wound site are useful for evaluating whether a given treatment is effective in an experimental setting but is not favored for a normal clinical setting.

[0065] As used herein, the term "isolated" when used in reference, for example, to platelets, means that the platelets are present at an enriched concentration relative to their concentration in circulating blood in vivo. Normal human blood has a platelet count of 150,000 to 400,000 per microliter. A sample is enriched if it has 800,000 platelets per microliter or more. The term encompasses platelet-rich plasma (PRP) with a concentration of 1 x 10⁶ platelets per microliter or more, including, for example, 1 x 10⁷ per microliter, 1 x 10⁸ per microliter, 2 x 10⁸ per microliter, or 3 x 10⁸ per microliter or more.

[0066] As used herein, the term "loaded" when used in reference to platelets means the platelets comprise an exogenous angiogenesis regulating factor. In this context it is emphasized that an "exogenous" angiogenesis regulating factor refers to an angiogenesis regulating factor that has been exogenously introduced to the platelets. Such exogenously added factors can include factors normally carried by platelets in vivo, e.g., VEGF, or factors not normally carried by platelets in vivo. Thus, the term "exogenous" as used in this context does not mean that the factor is not normally carried by platelets, but rather that the particular molecules of the factor were exogenously added to the platelets by those preparing a platelet preparation as described herein. It necessarily follows that platelets that have not been exposed to an exogenous source of angiogenesis regulating factor have not been "loaded" with that factor.

[0067] As used herein, the term "platelet activating agent" refers to an agent that stimulates platelets to release factors involved in the coagulation cascade and to secrete cytokines involved in wound healing and inflammation. Platelet activating agents include, but are not limited to collagen (which is exposed when the endothelial blood vessel lining is damaged), thrombin, (primarily through PAR-I), ADP, ADP receptors (P2Y1 and P2Y12) expressed on platelets, a negatively charged surface (e.g., glass), serotonin, acetylcholine and combinations thereof.

[0068] As used herein, the term "PAR-I agonist" refers to an agent that activates Protease Activated Receptor- 1 (thrombin receptor) signaling activity. PAR-I on platelets is activated by a mechanism involving proteolytic cleavage of a portion of the extracellular domain to generate a new N-terminus which then acts as a tethered or intramolecular ligand (agonist) for the receptor. The hexapeptide SFLLRN-NH2 comprising the new N-terminus after cleavage is referred to as the Thrombin Receptor Activating Peptide, or "TRAP." Peptide constructs based on this peptide sequence have strong PAR-I agonist activity. PAR-I agonists thus include, for example, TFLLR-NH₂ (Ma et al., 2004, PNAS 102: 216-220); TFLLRNPNDK; SFLLRNPNDKYEPF; and SFLLRN-NH_2. Similarly, agents that interfere with the binding of this peptide to the receptor tend to have antagonist activity.

[0069] As used herein, the term "PAR-4 antagonist" refers to an agent that antagonizes the activity of Protease Activated Receptor-4 (also a thrombin receptor) signaling activity. PAR- 4 on platelets is activated by a mechanism involving proteolytic cleavage of a portion of the extracellular domain to generate a new N-terminus which then acts as a tethered or intramolecular ligand (agonist) for the receptor. Examples of PAR-4 antagonists include transcinnamoyl- YPGKF-NH₂, (tcY-NH₂; Ma et al., 2005, Proc. Natl. Acad. Sci. U.S.A. 102: 216-220) and YD-3, a non-peptide PAR4 antagonist (ethyl 4-(l-benzyl-lH-indazol-3- yl)benzoate; see Wu & Teng, 2006, Eur. J. Pharmacol. 546: 142-147). BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the results of treatment of full thickness cutaneous wounds of diabetic mice with platelet preparations from healthy ("wild-type") or tumor-bearing animals. The figure shows the clinical aspects of wounds on day 10 post wound. Panel A shows full-thickness wounds excised on the dorsum of db/db mice and folio wed-up twice a week for four weeks. One will readily note the advanced healing status induced by Tumor Conditioned PRP (89% wound closure), when compared to both wild type PRP (63%) and wounds left to heal spontaneously (49%). Scale bar 0.5 cm.

Panel B shows time to 90% wound Closure. Tumor conditioned PRP treated wounds achieved 90% wound closure in a shorter time (11.1 days) when compared to both wild type PRP (16.7) and wounds left to heal spontaneously (20.6). **=p<0.01 compared to wt PRP and NT, *p<0.01 compared to NT. NT=Non-treated wounds, WT PRP=wild type platelet rich plasma treated wound (lOOμl), T PRP=tumor conditioned platelet rich plasma treated wounds (100 μl).

Figure 2 shows cell proliferation in wounds treated with wild-type PRP and PRP from tumor bearing animals (TPRP). Cross sectional wound tissues were harvested on day 10, stained for Ki67and photographs were taken from the middle of the wound bed (data not shown). Positively (e.g., brown) stained cells are actively proliferating and were quantified by counting (Figure T). Tumor conditioned PRP treated wounds showed on day 10 higher levels of proliferation (46.2%) when compared to both wild type PRP (23.2) and wounds left to heal spontaneously (20.2). *=p<0.01 compared to wt PRP and NT. NT=Non-treated wounds, WT PRP=wild type platelet rich plasma treated wound (lOOμl), T PRP=tumor conditioned platelet rich plasma treated wounds (100 μl).

Figure 3 shows quantified results of staining wound tissues for CD31. Cross sectional wound tissues were harvested on day 10. stained for CD31 (PECAM-I), and photographs were taken from the middle of the wound bed (data not shown). Microphotographs were also obtained from cross sectional wound tissues stained for phosphorylated VEGF Rec2/3 on day 10 (data not shown). Both PRP treatments induced activation of the VEGF pathway, as opposed to wounds left to heal spontaneously which did not show any staining (data not shown). Three pictures were taken in from each wound tissue (one in the middle and two from the edges of the wounds; data not shown) and blood vessels were quantified. Tumor conditioned PRP treated wounds showed higher number of blood vessels when compared to both wild type PRP (23.2) and wounds left to heal spontaneously (20.2). **=p<0.01 compared to wt PRP and NT, *p<0.01 compared to NT. NT=Non-treated wounds, WT PRP=wild type platelet rich plasma treated wound (lOOμl), T PRP=tumor conditioned platelet rich plasma treated wounds (100 μl).

Figure 4 shows a characterization of the proteome of platelets (A) and plasma (B) derived from tumor bearing and healthy mice. The angiogenic proteome was analyzed, showing a characteristic distribution between platelets and plasma. The majority of the angiogenic factors analyzed were found to have comparable levels in healthy and tumor conditioned samples.

Figure 5 shows the healing staging system; wound tissues on day 10 were staged using a two dimensional plot representing vascularity on the X-axis and cell proliferation on the Y-axis. NT was set to 1. T PRP induced the highest histological stimulation of the wound tissues when compared to both WT PRP and NT. Previously frozen TPRP and PRP failed to acheive the same levels of angiogenic stimulation of TPRP and PRP. Frozen TPRP kept some proliferative potential.

DETAILED DESCRIPTION

[0070] Methods and compositions for enhanced wound healing are provided herein. The methods and compositions take advantage of the ability of platelets to deliver angiogenesis regulating factors. In particular, the methods and compositions described herein take advantage of the ability of platelets to deliver exogenously added angiogenesis-regulating factors that aid the healing of wounds. This ability to deliver angiogenesis-regulating factors represents an advance in wound healing approaches because while the benefits of angiogenic factors for wound healing may be recognized, in many instances simply injecting angiogenic factors into a wound or into the circulation does not result in a therapeutic benefit. The proangiogenic factor VEGF, for example, is rapidly cleared from the circulation. The delivery of VEGF or other angiogenic factors to a wound site by platelets circumvents this problem and can provide a high local concentration of the factors directly to wound tissues.

[0071] Activated platelets have long been known to release granules containing factors involved in the coagulation cascade. The inventors have discovered, however, that platelets achieve the delivery of angiogenic factors not by simply secreting or "releasing" the factors, but rather, by extending processes from the platelet cell surface that contact the target endothelial cell. By doing so, there is no release of the factor to the general circulation, and thus little chance for the factors to be diluted out or otherwise cleared before they contact cell surface receptors on the target cell. The release of exogenously added factors by this mechanism from platelets placed at the wound site provides for the effective delivery of such factors directly to the wound site.

Preparation of Platelets:

a) Isolation of platelets

[0072] Platelets for use in the methods and compositions described herein can be isolated from a donor or from the individual to be treated with the composition. That is, platelets can be non-autologous or autologous. However, autologous platelets are preferred, primarily because they avoid possible infectious disease issues encountered with non-autologous donor blood products. Methods for the isolation of platelets are well known in the art.

[0073] In one aspect, autologous platelet isolation from whole blood can be performed using a device commercially designed for that purpose, e.g., the Magellan Autologous Platelet Separator (Medtronic Inc., Minneapolis, MN), according to the manufacturer's instructions. The device permits the isolation of platelet-rich plasma (PRP). The term "platelet-rich plasma" has a well known meaning in the medical arts, and its method of preparation is well established.

[0074] Preferably, autologous platelet-rich plasma is prepared by drawing blood from the individual having the wound, centrifuging the blood, and drawing off the supernatant (platelet-rich plasma), which comprises plasma, white blood cells, and platelets. The amount of platelet-rich plasma required understandably depends on the size of the wound to be treated. However, 10 mL of platelet-rich plasma is a convenient amount to obtain, requiring approximately 20 mL of blood to be drawn. Thus, 15 to 45 mL of blood is normally drawn in a conventional manner from the individual, and the blood is placed in a stopper top vacuum tube containing sodium citrate to prevent coagulation (available, e.g., from BD (Becton, Dickinson and Company) under the trademark "VACUT AINER").

[0075] The blood is centrifuged at speeds sufficient to produce forces of 135 to 280 g for 3 to 5 minutes at 20 to 37° C. The platelet-rich plasma is then drawn off to be used in the compositions and methods as described herein. Obviously, the same platelet isolation approach can be applied for the isolation of non-autologous platelets. Further concentration of PRP can be achieved by additional centrifugation steps. Final concentrations of platelets in PRP preparations can be adjusted, if necessary, using platelet free plasma remaining from the isolation process. A platelet concentration of , for example, 3 x 10⁸ platelets per microliter is readily achieved using these methods.

b) Addition of exogenous angiogenic factors to platelets

[0044] Exogenous angiogenic factors can be added to platelets in any of several different ways. Platelets are known to take up material from their surroundings, either actively by endocytosis or passively, e.g., by diffusion. A number of procedures have been suggested for the modulation of the granular content of platelets, but the simple exposure of platelets to media enriched with a specific protein is sufficient for "loading" proteins into platelet alpha- granules. First, one or more factors can be added to platelets by passive diffusion, i.e., the platelets are incubated with the angiogenic factor(s), e.g., at room temperature for 5 minutes or more. In one embodiment, it is preferred that the platelet loading approach not include, for example, exposure to liposomes or virus particles. Simple incubation of platelets with the exogenous factor results in the uptake of the factor by the platelets, including uptake into platelet alpha granules.

[0045] Alternatively, platelets can be loaded with an exogenous angiogenic factor by passing isolated platelets through a network with a diffusion barrier or membrane. In one aspect, on one side of the barrier is conditioned medium from cells that secrete one or more angiogenic factors, which pass through the barrier to where the platelets take them up. In another aspect, the barrier can separate cells expressing the angiogenic factor from the platelets. An apparatus can be set up in which platelets are flowed through a network of passages defined by the barrier. In this manner a steady state flow can be used to generate the platelets loaded with exogenous angiogenic factor. Cells that express angiogenic factor(s) can be, for example, tumor cells known to secrete a given factor or factors, or, alternatively, recombinant cells that express and secrete the factor into their medium.

[0046] As described in the Examples herein, platelets take up and deliver angiogenic factors secreted by tumors. Thus, platelets can be flowed by a bed of cultured tumor cells in order to load the platelets with the factors generated by the tumor cells. For this approach, it is preferable, but not absolutely required, that the tumor cells be cultured in serum-free medium.

[0047] Additional approaches include, for example, stimulating the exchange of granules by the platelets such that angiogenic factors are taken up, and tagging the angiogenic factor to a moiety that is bound by a platelet cell surface receptor.

c) Angiogenesis-regulating factors

[0048] There are a wide variety of angiogenic factors that can be used in the wound healing methods and compositions described herein. One of skill in the art can decide which angiogenesis regulator will be used in a given situation. Most often, an exogenous angiogenesis-promoting factor will be used in a platelet preparation used for wound healing. However, it is contemplated that angiogenesis inhibiting factors can be loaded into platelets along with angiogenesis promoting factors. One reason such an approach might be employed is that platelets have been shown to release both proangiogenic (e.g., VEGF) and antiangiogenic factors (e.g., endostatin). The release of these factors is apparently selectively controlled through the PAR receptors PAR-I and PAR-4 (see Ma et al., Proc. Natl. Acad. Sci. U.S.A. 102: 216-220). Specifically, PAR-I activation causes the release of proangiogenic factors, including VEGF, and the repression of endostatin release. PAR-4 activation causes the release of antiangiogenic factors, including endostatin, and the repression of VEGF release. This counter-regulatory mechanism may be effective in both starting the angiogenesis necessary for the healing process and in avoiding shutting down angiogenesis when the healing process is complete. Therefore, it is contemplated that it can be useful to load platelets with both angiogenesis promoting factors (proangiogenesis factors) and angiogenesis inhibiting factors (antiangiogenesis factors). In this instance, either the natural milieu of the wound environment would dictate which factors are released from the platelets at what time (e.g., proangiogenic factors early in the healing process, antiangiogenic factors later in the process), or additional factors, e.g., PAR agonists/antagonists, could be administered to the wound site to influence which factors are released by the loaded platelets at what time.

[0049] PAR agonists and antagonists are known in the art. For example, the hexapeptide SFLLRN-NH₂ and peptide constructs based on this peptide sequence have strong PAR-I agonist activity. PAR-I agonists thus include, for example, TFLLR-NH₂ (Ma et al., 2004, PNAS 102: 216-220); TFLLRNPNDK: SFLLRNPNDKYEPF; and SFLLRN-NH_2. Similarly, agents that interfere with the binding of the SFLLRN-NH₂ peptide to the PAR-I receptor tend to have antagonist activity.

[0050] Examples of PAR-4 antagonists include transcinnamoyl- YPGKF-NH₂, (tcY-NH₂; Ma et al., 2005, Proc. Natl. Acad. Sci. U.S.A. 102: 216-220) and YD-3, a non-peptide PAR4 antagonist (ethyl 4-(l-benzyl-lH-indazol-3-yl)benzoate; see Wu & Teng, 2006, Eur. J. Pharmacol. 546: 142-147).

[0051] Angiogenesis promoting factors that can be used in compositions and methods described herein include, for example:

Angiogenin;

Angiopoietin-1; oc2-antiplasmin; aFGF;

B61 (ligand for Eck receptor tyrosine kinase); bFGF;

DeI-I;

EGF;

EGFR;

FasL;

Follistatin;

Granulocyte colony- stimulating factor (G-CSF);

Heparin sulfate proteoglycan;

Hepatocyte growth factor (HGF)/Scatter factor (SF);

12-hydrozyeicosatetraenoic acid;

IGF-I;

IGFR;

Interleukin-8 (IL-8);

Leptin; oc2-macro globulin;

Midkine ;

PAR-I agonist;

PAR4-antagonist;

PDGF;

Platelet-derived endothelial cell growth factor (PD-ECGF);

Platelet-derived growth factor-BB (PDGF-BB);

PDGFR;

PF-4;

Placental growth factor 1 and 2;

Plasminogen activator inhibitor- 1 (PAIl);

Pleiotrophin (PTN);

Progranulin;

Proliferin;

Sema-E; Sema-III;

Sema-IV,

Soluble vascular cell adhesion molecule- 1;

Soluble E-selectin;

Tat protein of HIV-I;

TGF-α;

TGF-βl;

Thrombospondin-1 (Tspl);

TNF-α;

VEGF-A;

VEGF-B;

VEGF-C;

VEGF-D and other factors that activate the VEGF signalling pathways (including, but not limited to VEGF121, VEGF145, VEGF165, VEGF168, VEGF189; Agents that activate the neuropilin 1 & 2 pathways; and VEGF receptors, e.g., VEGFRl (flt-1), VEGFR2 (flk-2), VEGFR3 (flt-4);

[0052] Combinations of these or other proangiogenic factors can also be used in the methods and compositions described herein.

[0053] Angiogenesis inhibiting factors that can be used in compositions and methods described herein include, for example:

Angioarrestin;

Angiostatin (plasminogen fragment);

Antiangiogenic antithrombin III;

Cartilage-derived inhibitor (CDI);

CD59 complement fragment;

Endostatin (collagen XVIII fragment);

Fibronectin fragment;

Gro-beta;

Heparinases;

Heparin hexasaccharide fragment;

Human chorionic gonadotropin (hCG);

Interferon alpha/beta/gamma;

Interferon inducible protein (IP-10);

Interleukin-12;

Kringle 5 (plasminogen fragment);

Metalloproteinase inhibitors (TIMPs);

2-Methoxyestradiol;

Placental ribonuclease inhibitor;

Plasminogen activator inhibitor;

PF4;

Prolactin 16kD fragment;

Proliferin-related protein (PRP);

Retinoids; Tetrahydrocortisol-S ; Thrombospondin-1 (TSP-I); Transforming growth factor-beta (TGF-β); Vasculostatin; and Vasostatin (calreticulin fragment)

[0054] Combinations of these or other antiangiogenic factors can also be used in the methods and compositions described herein. It is acknowledged that several of the antiangiogenic factors are also listed with the proangio genie factors. This is a reflection of the fact that some factors can participate in both pro- and anti-angiogenic processes, depending upon their context, and indeed, upon when in the healing or angiogenic process they are used. This does not indicate unpredictability in the process, but rather points to the fine-tuned control of the angiogenic process in vivo. If any doubt remains as to whether a given factor is pro- or antiangiogenic in a given set of circumstances, one of skill in the art can determine with a minimum of experimentation which activity that factor has in the context of the wound healing methods and compositions described herein - that is, when a given factor is involved, it requires only routine experimentation, e.g., in an animal model system as described herein, to test the given factor for its effect in a wound healing method or composition as described herein.

[0055] Functional fragments of known angiogenesis regulating factors are also contemplated for use in the methods and compositions described herein. By "functional fragment" is meant a fragment that substantially retains the angiogenesis regulating activity of the full length factor ("substantially retains" means the fragment retains at least 80% of the activity of the full length factor). Guidance as to fragments that will continue to bind a given receptor can be found in the crystal structure of the receptor. A large number of receptors have been crystallized, frequently in complex with corresponding ligands. Such crystal structure information provides guidance as to exactly what regions of a ligand interact with the receptor, and provide information that permit the in silico screening/modeling of compounds expected to bind and activate (or inhibit) the receptor. That is, the crystal structure can provide structure-function correlations that permit one to readily grasp the types of ligands that will have similar binding activity for a given receptor. As one example, where an angiogenesis-regulating factor influences the well known VEGF-mediated angiogenesis pathway, the structure of the VEGF ligand or its receptor(s) involved can be instructive in understanding, for example, what fragments or variants of the ligand would be expected to have similar activity on the receptor. The crystal structure of VEGF at 2.5 A is reported by Muller et al., 1997, Proc. Natl. Acad. Sci. U.S.A. 94: 7192-7197. Those authors report that VEGF is a homodimeric member of the cystine knot family of growth factors, with limited sequence homology to platelet-derived growth factor (PDGF) and transforming growth factor p2 (TGF-p). They determined the crystal structure at 2.5 A resolution, and identified its kinase domain receptor (KDR) binding site using mutational analysis. The reference teaches that the VEGF monomer resembles that of PDGF, but its N-terminal segment is helical rather than extended. The dimerization mode of VEGF is taught to be similar to that of PDGF and very different from that of TGF-β. Mutational analysis of VEGF revealed that symmetrical binding sites for KDR are located at each pole of the VEGF homodimer. Each site was found to contain two functional "hot spots" composed of binding determinants presented across the subunit interface. What were classed as the two most important determinants are located within the largest hot spot on a short, three- stranded sheet that is conserved in PDGF and TGF-β. Functional analysis of the binding epitopes for two receptor-blocking antibodies reveal different binding determinants near each of the KDR binding hot spots.

[0056] The structure/function correlations for the VEGF- VEGF-receptor system are described, for example, by Shibuya, 2001, Cell Structure and Function 26: 25-35. Structurally, VEGF belongs to the VEGF-PDGF super-gene family. In this gene family, 8 cysteines are conserved at the same positions. Two of these sites form intermolecular cross linking S-S bonds to form a dimer, while the other six sites form intramolecular S-S bonds to form 3 loop structures. In humans, VEGF has at least three important sub-types, of 121, 165 and 189 amino acids, generated by alternative splicing. The 121 and 165 amino acid subtypes, VEGF₁₂₁ and VEGF_16S are representative forms, with VEGF_16S bearing an additional 44 amino acid basic stretch relative to VEGF₁₂₁. Through the basic stretch, the VEGF₁₆₅ molecule binds heparin or heparin-like molecules in the matrix and on the cell surface, and can also associate with the cell surface molecule neuropilin-1. The association of VEGF with neuropilin-1 has been reported to increase the affinity of VEGF_16S with one of the VEGF receptors, KDR (VEGFR2), about 10-fold, such that VEGF₁₆₅ is the strongest signal transducer of the naturally-occurring VEGF subtypes.

Angio genesis Assays:

[0057] In general, and as discussed above, one of skill in the art will know whether a given agent or factor is an angiogenesis-regulating factor prior to loading platelets with such agent. For the avoidance of doubt, one can use any of a number of in vitro or in vivo angiogenesis assays to evaluate the influence of a given factor or agent on angiogenesis.

The CAM assay:

[0058] The chick chorioallantoic membrane (CAM) assay is frequently used to evaluate the effects of angiogenesis regulating factors because it is relatively easy and provides relatively rapid results. An angiogenesis regulating factor useful in the methods and compositions described herein will modify the number of microvessels in the modified CAM assay described by Iruela-Arispe et al., 1999, Circulation 100: 1423-1431. The method is based on the vertical growth of new capillary vessels into a collagen gel pellet placed on the CAM. In the assay as described by Iruela-Arispe et al., the collagen gel is supplemented with an angiogenic factor such as FGF-2 (50 ng/gel) or VEGF (250 ng/gel) in the presence or absence of test proteins/peptides. To test whether a factor promotes angiogenesis, the FGF-2 or VEGF in the collagen gel can be omitted or reduced, and replaced or supplemented with the factor under examination. The extent of the angiogenic response is measured using FITC- dextran (50 μg/mL) (Sigma) injected into the circulation of the CAM. The degree of fluorescence intensity parallels variations in capillary density; the linearity of this correlation can be observed with a range of capillaries between 5 and 540. Morphometric analyses are performed, for example, by acquisition of images with a CCD camera. Images are then imported into an analysis package, e.g., NHImage 1.59, and measurements of fluorescence intensity are obtained as positive pixels. Each data point is compared with its own positive and negative controls present in the same CAM and interpreted as a percentage of inhibition, considering the positive control to be 100% (VEGF or FGF-2 alone) and the negative control (vehicle alone) 0%. Similar evaluations are performed for positive regulators of angiogenesis, relative to controls lacking such an agent or factor. Statistical evaluation of the data is performed to check whether groups differ significantly from random, e.g., by analysis of contingency with Yates' correction.

Additional Angiogenesis Assays:

[0045] Additional angiogenesis assays are known in the art and can be used to evaluate factors for use in the methods and compositions described herein. These include, for example, the corneal micropocket assay, hamster cheek pouch assay, the Matrigel assay, hindlimb ischemia assay, and modifications thereof, and co-culture assays. Donovan et al. describe a comparison of three different in vitro assays developed to evaluate angiogenesis regulators in a human background (Donovan et al., 2001, Angiogenesis 4: 113-121, incorporated herein by reference). Briefly, the assays examined include: 1) a basic Matrigel assay in which low passage human endothelial cells (Human umbilical vein endothelial cells, HUVEC) are plated in wells coated with Matrigel (Becton Dickinson, Cedex, France) with or without angiogenesis regulator(s); 2) a similar Matrigel assay using "growth factor reduced" or GFR Matrigel; and 3) a co-culture assay in which primary human fibroblasts and HUVEC are co- cultured with or without additional angiogenesis regulator(s) - the fibroblasts produce extracellular matrix and other factors that support HUVEC differentiation and tubule formation. In the Donovan et al. paper the co-culture assay provided microvessel networks that most closely resembled microvessel networks in vivo. However, the basic Matrigel assay and the GFR Matrigel assay can also be used by one of skill in the art to evaluate whether a given factor is an angiogenesis-regulating factor as necessary for the methods and compositions described herein. Finally, an in vitro angiogenesis assay kit is marketed by Chemicon (Millipore). The Fibrin Gel In Vitro Angiogenesis Assay Kit is Chemicon Catalog No. ECM630.

Platelet wound healing compositions:

[0046] The treatment methods described herein use platelet wound healing compositions comprising platelets bearing exogenously added angiogenesis regulating factors. In their simplest form, the platelets, loaded with exogenous factor(s), can be formulated in high concentration liquid suspension, e.g., at 1-5 x 10⁸ platelets per microliter in plasma. This preparation can be overlaid onto a wound and covered with a dressing (preferably a non- absorbent dressing) to enhance the healing of the wound.

[0047] In particular embodiments, the platelet compositions useful in the methods and compositions described herein can additionally comprise additional carriers or excipients as known in the art for topical administration. The term "topical administration" is used in its broad sense to refer to administration to a wound surface. That surface can be internal or external. It is contemplated that in other particular instances it can be beneficial to administer platelet compositions with a minimum of additional material, i.e., without gelling agent or other excipients. [0048] In another aspect, a gel can be prepared using the loaded platelets, and the gel is the form that is administered to the wound surface. Platelet gels, and particularly autologous platelet gels are well known in the art. Loaded platelet preparations as described herein can be formulated as gels by exposing the platelets to calcium and a platelet activating agent, e.g., thrombin, collagen, serotonin, ADP, acetylcholine and combinations thereof. Naturally, the same approaches used to generate autologous platelet gels can be used to prepare platelet gels for non-autologous application if so desired.

[0049] Platelet gel formulations are described, for example, in U.S. Patent No. 7,112,342 (which describes, among other things, a platelet gel formulation including platelets, calcium, thrombin and an anti-oxidant, which slows the gel formation and prevents the formation of a hard platelet gel mass), 6,841,170, 6,942,880 and in published U.S. Patent Applications 20020172666, 20030198687 and 20060095121, each of which is incorporated herein by reference. Further, Horn et al. describes the preparation and use of autologous platelet gel on acute human skin wounds (Horn et al., 2007, Arch. Facial Plast. Surg. 9: 174-183, which is incorporated herein by reference).

[0050] Commercial products for the preparation of platelet gels, including autologous platelet gels include, for example, autologous platelet gel products provided by Blood Recovery Systems, Inc., the Symphony™ II platelet concentrate system from DePuy Orthopaedics, Inc., and the Symphony™ PCS platelet concentrate system, also from DePuy Orthopaedics, Inc.

[0051] In one embodiment, a platelet preparation of use in the methods and compositions described herein is formed into a semi-solid gel or paste by combination of loaded platelets with, for example, a cellulose or other exogenous gelling agent. Collagen gel, for example, would likely form a gel with platelets, in part because exposure of platelets to collagen in vivo activates the platelets. However, in order to maintain the regulated release characteristic of platelets in vivo, it may be preferred that the platelets not be activated prior to exposure to the wound. In this instance, it would be preferred that the platelet formulation not comprise collagen or a collagen gel, and it would be preferred that no platelet activating agent be added to the platelet preparation. In such preparations, platelet activation can still occur at the wound site, for example, when the loaded platelets come in contact with wound associated activators, including, e.g., endogenous thrombin, collagen, etc. Similarly, many methods have been used to enhance platelet activation (change in temperature, ions, specific molecules, such as thrombin, ADP and N- Acetyl- Glucosamine)⁴²'⁴³ but it has been found that the simple exposure of affected tissues may provide sufficient stimulus for platelet activity (see the Examples, below). In some embodiments, then, no platelet activation agent is used in platelet preparations as described herein. In other embodiments, it is preferred that no gelling agent is used with the platelet preparation.

[0052] The platelet compositions described herein can be administered in conjunction with other agents or treatments for enhanced wound healing. These include, but are not limited to, for example, extracellular matrix analogs (e.g., Integra (Integra LifeSciences, Plainsboro, NJ), AlloDerm (Lifecell, Inc., Branchburg, NJ), etc.), V.A.C.™ negative pressure wound therapy approaches (KCI, Inc.), cultured epithelial autografts, tissue engineered products and topical growth factors.

Use of Platelet Compositions

[0053] Platelet compositions as described herein are useful to enhance or aid in wound healing. In practice, the preparations described are placed in contact with a wound surface (including an internal or external wound surface) in order to enhance the healing of the wound. A dressing can be applied to maintain the platelet composition in place. Where the wound is an internal wound, the wound healing platelet composition can include, for example, an occlusive dressing that prevents the composition from diffusing. Such dressings are known to those of skill in the art and include dressings or preparations that are absorbed by the body over time. Alternatively, or in addition, where the compositions are applied to internal wounds, extracellular matrix products such as Integra™ can be employed to assist in retaining the platelet composition in the desired location.

Wounds

[0054] The methods and compositions described herein can be used to treat a number of different types of wounds. One application of particular importance is enhancing the healing of poorly healing or chronic wounds, such as diabetic wounds and decubitus ulcers, among others. Such wounds have been known to remain for weeks, months or even years, and present an ongoing risk of infection for the entire period in which an open wound remains.

[0055] In addition to diabetic wounds and decubitus ulcers, wounds that can be treated using the methods and compositions described herein include, for example, any chronic or poorly healing wound, venous stasis wounds, vascular wounds, radiation wounds, steroid wounds, acute surgical wounds, burns, and traumatic lacerations. Additional uses of wound healing compositions as described herein include, for example, sealing down flaps to avoid seromas, bolstering anastamoses of bowel, urinary system and blood vessels, repair of bronchial and tracheal defects, closure of the dura, repair of leaks, e.g., repair of CSF leaks, bile leaks, pancreatic leaks, etc., and repair of bony defects.

[0056] The ability to manipulate the angiogenic factor profile of platelets is also recognized as providing a method of inhibiting angiogenesis where so desired. Thus, antiangiogenic factors can be loaded and the platelets administered for the treatment of, for example, hypertrophic granulation tissues, vascular malformations, pyogenic granuloma and tumors.

[0057] The platelet compositions described herein can be administered in a range of frequencies, that will vary with the type of wound being treated and the exact formulation of the composition. Compositions can be administered, for example, once when an internal wound surface is to be treated, prior to suturing or otherwise closing the external access to the internal wound surface. For other wounds, e.g., those on an external surface or on an internal surface accessible with non-surgical approaches (e,g., surfaces in the mouth, colon, vagina, esophagus, trachea, nasal passages, etc.), application can be more frequent, e.g., an initial application, followed by re- application within hours, e.g., 4 hours, 8 hours, 12 hours, etc., or, more likely, followed by re- application once or twice daily, for example, until the wound is closed. In practicality, any range of re- application that maintains the rate of healing can be used by the ordinarily skilled practitioner.

[0058] Dosages of platelets bearing exogenously added angiogenesis regulating factors will also vary with the type and size of the wound. Clearly, a larger wound surface will benefit from a larger amount of a wound healing composition, relative to a smaller wound surface. The concentration of platelets in the compositions administered can also vary, but can generally be on the order of 5 x 10⁷ platelets per cubic millimeter of the composition or higher, e.g., 1 x 10⁸ platelets per mm³, 1.5 x 10⁸ platelets per mm³, 2 x 10⁸ platelets per mm³, 2.5 x 10⁸ platelets per mm³, 3 x 10⁸ platelets per mm³, 3.5 x 10⁸ platelets per mm³, 4 x 10⁸ platelets per mm³ or higher.

[0059] Efficacy of treatment can be judged by an ordinarily skilled practitioner. Clearly, where a chronic wound is involved, any healing that leads to closure of the wound involves effective treatment. Alternatively, where the wound is not a chronic wound, e.g., an acute surgical wound, changes in the time required to close the wound (i.e., in the rate of healing) will be apparent to the ordinarily experienced practitioner based on their frequent experience with similar wounds.

[0060] Efficacy for any given formulation (e.g., platelets loaded with or bearing a given exogenous angiogenesis regulating factor and in a given delivery formulation) can also be judged using an experimental animal wound healing system, e.g., wild-type mice or rats, or preferably, a diabetic mouse system akin to that described in the Examples herein below. When using an experimental animal model, efficacy of treatment is evidenced when wound closure for a standardized wound occurs earlier in treated, versus untreated wounds. By "earlier" is meant that wound closure occurs at least 5% earlier, but preferably more, e.g., one day earlier, two days earlier, 3 days earlier, or more.

Kits:

[0061] Also provided by the instant disclosure are kits for the preparation of platelet compositions as described herein. Kits can include, for example, one or more angiogenesis regulating factors and either a platelet preparation or reagents and containers necessary for the preparation of a platelet preparation. For example, a kit for the preparation of a platelet composition can include an angiogenesis-regulating factor (e.g., VEGF), and vacuum tubes comprising citrate anticoagulant for the isolation of blood to be used for the isolation of platelets. Such a kit can additionally comprise buffers or reagents and tubes for the successive centrifuge spins used to concentrate and purify platelets from whole blood and/or a container(s) to hold the platelet composition once prepared. A kit can also comprise a medium for the incubation with exogenous angiogenesis regulating factor (although plasma will often be the medium). A kit can further comprise any agent to be combined with the loaded platelet composition to formulate it for a particular type of administration or delivery - e.g., cellulose or other thickening agent, or calcium and a platelet activating agent, e.g., thrombin.

EXAMPLES Example 1. Evaluation of Tumor-Conditioned Platelets [0062] In this study tumor-conditioned platelets (TCPs); i.e., platelets derived from tumor- bearing mice, were tested for their effect on the process of wound healing. Unprecedented findings were made that tumor-conditioned platelets considerably benefit wound healing. With the increase in the prevalence of diabetes type 2 and its complications, numerous chronic wounds can be amenable to therapies involving such platelets, or platelets engineered to carry exogenous angiogenesis-regulating factors.

Materials & Methods:

In vivo preparation of TCPs

[0063] Wild-type, male, C57BL/6 male mice were injected subcutaneously with 1 x 10⁶ Lewis lung carcinoma (LLC) cells in the dorsal flanks or received a sham injection of sterile water. After 4-6 weeks or once the mice began showing signs of wasting and lethargy (nodule was palpated at the injection side), they were euthanized and their blood was collected by a terminal bleed via heart puncture. Platelet-rich plasma (PRP) was obtained through a series of centrifugations as previously described and the platelet concentration was adjusted to 3 x 10⁸ / μl. After collection, 100 μl of freshly-obtained tumor-conditioned PRP, which is designated henceforth as tumor-conditioned platelets (TCPs), was applied topically as described below in a blinded fashion.

Wound Model & Study Design

[0064] Homozygous, genetically diabetic, 10 week-old Lep/r - db/db male mice (strain C57BL/KsJ-Lepr^db) were used under an approved animal protocol in an association for assessment and accreditation of laboratory animal care international (AAALAC) accredited facility. One day prior to surgery, all mice were shaved and depilated (Nair^®, Church & Dwight Co., Princeton, NJ). On the day of the surgery, animals were weighed and anesthetized with 60 mg/kg Nembutal (Pentobarbital, Abbott Laboratories, Chicago, II) and prepared to receive one dorsal wound. Full-thickness, 1.0 cm areas of skin and panniculus carnosus were excised, and the wounds were photographed prior to treatment. Wounds were individually sealed with semi-occlusive polyurethane dressings (Tegaderm™, 3M, St. Paul, MN), and TCPs (lOOμl at ~3 x 10⁸ platelets/μl) were injected into randomly- selected wounds through the dressing using a 30 gauge needle. Groups were categorized and compared as follows: 1. Wounds left untreated as negative controls (NT, n=12); 2. Wounds treated with wild type PRP (WT PRP, n=12) and 3. Wounds treated with tumor conditioned PRP (T PRP, n=12). On days 10 and 21, 6-8 animals per group were euthanized and the wounds were photographed for macroscopic morphology, excised and fixed in 10% neutral-buffered formalin solution.

Wound Closure Analysis

[0065] Digital photographs captured twice a week were compared using planimetric methods with initial photographs taken at day 0 by three independent observers who were blinded to the treatment mode. Wound closure was quantified by measuring contraction (C), re- epithelialization (E) and open wound (O) as a percentage of the original wound area. The sum of contracted, re-epithelialized and open wound areas equals 100% of the original wound size.¹⁷

[0066] Central wound cross-sections were embedded in paraffin, sectioned, and stained according to routine Hematoxylin and Eosin (H&E) protocols. Panoramic cross-sectional digital images of each wound were prepared using Adobe Photoshop CS Software (Adobe Systems Incorporated, San Jose, CA) to analyze granulation tissue area and thickness with digital planimetry (Image J, NIH, Bethesda, MD).

(1) Immunohistochemistry

[0067] Paraffin-embedded sections were re-hydrated followed by antigen retrieval of Ki67 (antigen located on the surface of the chromosomes) (10 min microwave in 10 mM sodium citrate (pH 6.0)) and for platelet endothelial cell adhesion molecule one (PECAM-I) (waterbath at 37⁰C in proteinase K solution (14-22 mg/ml)). Sections were incubated with primary antibodies to PECAM-I (Pharmingen, San Jose, CA) and Ki-67 (Lab Vision, Freemont, Ca) at 4°C overnight and 1 hour at room temperature, respectively. PECAM-I signal was intensified using tyramide amplification system (Perkin Elmer, Boston, MA). The total number of PECAM-I positive blood vessels was counted in each high power field using 4Ox magnification. Similarly, a ratio of proliferating nuclei (positive for Ki-67) over total nuclei was quantified at 4Ox magnification to establish proliferative index. For each treatment arm and immunolocalization condition three microscopic fields per wound (n=6 wounds per group), two from the edges and one from the middle of the wound, were evaluated at 4Ox magnification. Platelet and plasma preparation for SELDI-ToF MS

[0068] Platelet pellets were separated from the corresponding platelet poor plasma (PPP) from PRP and TPRP preparations by an additional centrifugation step at 90Og at room temperature. The two preparates were then processed and analyzed by SELDI-ToF technology (Ciphergen, Fremont, California). Platelet pellets and 20 μL of PPP from each mouse were lysed using 25 μL and 40μL, respectively, of U9 buffer (2% CHAPS (3-[(3- cholamidopropyl) dimethylammonio]-l-propansulfonate), 50 mM Tris-HCl, pH 9 (Ciphergen) for 1 hr at room temperature. Platelet lysates were then centrifuged at 10,000 g for 1 min at 4⁰C. Both platelet extracts and PPP were fractionated by anion-exchange chromatography modified after the Expression Difference Mapping (EDM) Serum Fractionation protocol (Ciphergen, Fremont, CA). The fractionation was performed in a 96- well format filter plate on a Beckman Biomek® 2000 Laboratory Work Station equipped with a DPC® Micromix 5 shaker. An aliquot of 20 μL of the platelet and 60 μL of denatured plasma diluted with 100 μL of 50 mM Tris-HCl pH9 was transferred to a filter bottom 96- well microplate pre-filled with BioSepra Q Ceramic HyperD F sorbent beads re-hydrated and pre-equilibrated with 50 mM Tris-HCl, pH 9. All liquids were removed from the filtration plate using a multiscreen vacuum manifold (Millipore, Bedford, MA) into respective wells of 96-well microtitre plates with the capture of the initial flow-through as fraction 1. This step was repeated for subsequent incubations with 2 x 100 μL of the following buffers: pHs 7 (5OmM HEPES), 5 (5OmM NaAcetate), 4 (5OmM NaAcetate), 3 (5OmM NaCitrate) (fractions 2, 3, 4 and 5, respectively), followed by a final organic wash (33% isopropanol/16.7% acetonitrile (ACN)/0.1% trifluoroacetic acid (TFA)). Expression difference mapping (EDM) on ProteinChip arrays was carried out using weak cationic exchange chromatography protein arrays (CMlO ProteinChip arrays; Ciphergen, Fremont, CA) by loading sample fractions onto a 96-well bioprocessor, and equilibrating with 50 mM sodium acetate (Sigma, St. Louis, MO), pH 4. A further dilution of 40 μL anion exchange chromatography fraction into 100 μL of the same buffer on each array spot was incubated for 1 hr. Array spots were washed for 3 minutes with 100 μL 50 mM sodium acetate, pH 4. After rinsing with water, 2 x 1 μL of sinapinic acid matrix solution was added to each array spot. For protein profiling, all fractions were diluted 1:2.5 in their respective buffers used to preequilibrate ProteinChip arrays. This step was followed by readings using the Protein Ciphergen System 4000 (PCS4000) SELDI- ToF mass spectrometer (Ciphergen, Fremont, CA) and processed with the ProteinChip Software Biomarker Edition™, Version 3.2.0 (Ciphergen, Fremont, CA). After baseline subtraction, spectra were normalized by means of a total ion current method. Peak detection was performed by using Biomarker Wizard software (Ciphergen, Fremont, CA) employing a signal-to-noise ratio of 3.

Wound Watch Staging System: Relative Quantification of Vascular and Proliferative compartments

[0069] To compare the healing under different treatment conditions, high power images of stained sections were used to quantify the degree of proliferation and vascularization. Three digital images of PECAM-I and Ki-67 stained slides were captured for each sample, one in the middle and two on the edges of the wound bed. Pictures were viewed with Adobe Photoshop CS Software, and blood vessels and proliferating nuclei in each high-powered field were marked and counted and Ki-67-positive cells were expressed as a ratio of proliferating nuclei to total nuclei. Ratios were calculated between results obtained from the centre of the lesions and from the edges of each treatment group to the plasma treated group. Fifteen microscopic fields were evaluated at 4Ox magnification for each experimental treatment.

Vessel Density Quantification

[0070] Digital color images of the wound sections were preprocessed before quantification to ensure uniform contrast of PECAM-I⁺ areas relative to the background. A mask of positive staining was created using the color mask function of the program CorelPhotoPaint v.lO by sampling five different chromogen color tones represented in positively-stained areas. The masked vessel areas were converted to pure black while the background was made pure white. Both regions were used for blood vessel quantification in IP Lab software by applying the segmentation function. Tissue regions were defined by projecting the original H&E image over the processed image. Blood vessel density, quantified over the entire image, was expressed as the ratio of vessel area to total granulation tissue area. Between 4 and 7 microscopic fields at 4Ox magnification were used to evaluate vessel density for each wound and treatment modality. Quantification of Cell Proliferation

[0071] Wounds were analyzed for cellular proliferation using image analysis of Ki67-stained sections in a manner similar to the method of vessel density quantification. High-powered digital images of Ki67-stained wound sections were used to measure the number of Ki67⁺ cells relative to the total number of nuclei (Fig. 2). The degree of proliferation was quantified over the entire wound section using 4-6 fields at 2Ox magnification and expressed as a ratio of proliferating nuclei (Ki67⁺) to total nuclei.

Statistical Analysis

[0072] Values were expressed as means + standard deviation in the text and figures. Oneway analysis of variance and ad hoc LSD tests were used to determine the significance of differences among treatments. Multivariate analysis was performed using Statistica v7.0 (StatSoft, Inc, Tulsa, OK).

Results:

T and WT PRP modulate Wound-healing kinetics.

[0073] Wounds treated with tumor-conditioned PRP exhibited a tendency toward faster wound closure starting from the first week of the follow up. T PRP induced a 1.8-fold increase (p<0.01) in wound closure when compared to NT wounds (Fig. IA) on day 10. WT PRP treatment showed an intermediate behavior between T PRP and NT (Fig IA).

[0074] T PRP treated wounds reached 90% wound closure in 11.1 days, reducing time to 90% closure by 5.6 and 9.5 days when compared to WT PRP and NT wounds, respectively. WT PRP treatment induced faster wound closure when compared to NT (Fig. IB, p<0.01).

Wound healing staging system.

[0075] Wounds left untreated in this animal model reached 50% wound closure approximately between day 7 and day 14. After two weeks of follow-up differences in wound healing between the groups tended to reduce, as the natural tendency of this rodent model to heal spontaneously prevaricated the effects induced by the treatments. Thus, day 10 was selected for staging wound healing. [0076] T PRP treated wounds reached higher levels of cell proliferation, as assessed by Ki-67 staining, a marker for proliferating cells. While the average percentage of proliferating cells in NT and WT PRP treated wounds were 20.2, and 23.2, respectively, T PRP treated wounds reached 46.2% (Fig. 2; p<0.01) on day 10.

[0077] When inflammatory infiltrate was analyzed, by staining wound section for CD45, a pan leukocytic marker, it was possible to notice that both T and WT PRP increased the inflammatory infiltrate in the wound bed on day 10 when compared to NT wounds (data not shown).

[0078] Wound bed vascularity was assessed by counting PECAM-I positive (a pan- endothelial marker) blood vessels in high power field. Wounds healing spontaneously (NT group) in the diabetic mouse model, showed lower levels of blood vessel formation on day 9, when compared to both T and WT PRP (Fig. 3). T PRP treatment increased vascularity also when compared to WT PRP (Fig 3). Immunohistochemistry with PECAM-I (a pan- endothelial marker) showed vascularization averaging 61 blood vessels per hpf in TPRP treated wounds, 44 vessels per hpf in PRP treated wounds.

[0079] The staining for the phosphorylated isoforms of VEGF Receptors 2/3 revealed higher levels of positivity when both T and WT PRP were compared to NT wounds, without differences between the two PRP treated groups. TPRP and PRP markedly increased VEGFR- 2/3 phosphorylation in the wound bed in comparison to untreated wounds, displaying a similar gradient of intensity that peaked in TPRP treated wounds (data not shown). By day 21, while wounds treated with TPRP and PRP demonstrated decreasing numbers of vessels compared to day 10, NT wounds showed an opposite trend (data not shown). Among all proliferating cells in the granulation tissue, several endothelial-like cells were found to be positive in the TPRP wounds (data not shown).

Wound collagen matrix deposition

Both PRP treatments significantly stimulated collagen deposition, when wounds were evaluated on day 21 with Mas son's Trichrome staining.

[0080] Diabetic wounds left untreated showed limited ability to form a collagenous wound matrix, exhibiting a friable and not homogeneous collagen bed. [0081] WT PRP treatment increased the amount and density of collagen, resembling an early stage of a scar tissue. T PRP treated wounds induced a unique pattern of distribution of the newly deposited collagen that was found in thick bundles alternating with area of absent collagen and high cellularity. PRP treated wounds exhibited a collagenous matrix fibers parallely oriented and relatively acellular, such as in any typical early scar tissue (data not shown).

[0082] In summary then, single, lcm dorsal, full-thickness wounds, on db/db mice were treated with T PRP, wild-type PRP (WT PRP) or left untreated (NT) and wound closure was followed up for four weeks. A single dose of T PRP (lOOμl, 3 x 10⁸platelets/μl) accelerated wound closure time, and cell proliferation (2.3-fold increase) compared to non-treated and WT PRP treated wounds on day 10. Both T PRP and WT PRP treatments increased wound tissue vascularity compared to non-treated wounds. On day 21, wounds left to heal spontaneously in the diabetic mouse show irregularly deposited collagen, and friable wound tissue matrix. Wounds treated with wild type PRP exhibited increased amount and organization of newly formed collagenous matrix, while tumor conditioned PRP treated wounds showed characteristic collagen bundles more focally distributed. T PRP stimulated healing across clinically-relevant parameters. Naturally conditioned or engineered platelets represent an innovative strategy for treating the increasing number of recalcitrant wounds. That is, wound healing compositions can be prepared, for example, from the platelets of individuals carrying tumors. While this is the case, given the ethical concerns of using material from tumor-bearing humans for therapy of others, a more palatable and practical approach is to load platelets of non-tumor bearing individuals with angiogenic factors, including angiogenic factors carried by platelets in tumor bearing individuals.

Growth factor analysis of platelets and plasma from healthy and tumor-bearing animals.

[0083] To determine whether the enhancement of angiogenesis by TPRP was due to increased levels of angiogenesis regulators, or whether it was due to an improved presentation of these regulators to the tissues we separated PRP and TPRP into their respective platelet and plasma components and submitted the samples to complete proteomic analysis using SELDI ToF technology. A number of previously identified angiogenesis regulators in platelets identified by the same technology in earlier studies was analyzed. Some of these previously identified proteins confirmed to be present in platelets in early tumor development are nerve growth factor (NGF), connective tissue activating protein-III (CTAPIII), platelet factor-4 (PF-4), β2-microglobulin, basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF) and vascular endothelial growth factor (VEGF). The proteomic analysis of platelets and plasma preparations from non-tumor-bearing and tumor- bearing mice did not reveal any elevations in these previously identified proteins, even though both platelets and plasma showed trends in the distribution of CTAPIII and PF-4, that were similar to those established for tumor growth⁵⁰.

Improved growth factor presentation by platelets of TPRP

[0084] To explore the hypothesis that the enhanced healing is due to an improved bioavailability of angiogenesis regulators presented by platelets, the ability of intact platelets to deliver and present angiogenesis related proteins to the wound bed was tested. While growth factors are preserved through freezing and thawing ¹³'⁵², platelets are subject to irreversible activation (degranulation) and loss of function ⁵³. An aliquot was frozen from each of the PRP preparations to control for the function of intact platelets. Previously frozen T PRP and PRP almost completely lost the ability to form blood vessels, even though the levels of proliferation induced by frozen T PRP were comparable to those found in wounds treated with PRP from non-tumor-bearing mice. Frozen PRP showed an almost complete decay of stimulatory function. To better conceptualize and compare the effects of the studied treatments, a 2D staging system was developed in which vascularity was plotted against cell proliferation⁵¹. The data presented in Figure 5 are normalized to the histological stage of NT wounds, which is given an arbitrary value of 1. TPRP treatment of wounds resulted in the highest histological stimulation of tissue repair as compared to NT wounds. TPRP induced over 4-fold increase (p<0.01, compared to both PRP and NT) in cell proliferation, while PRP induced a ~2.5-fold increase (p<0.05), compared to NT controls.

[0085] This Example presents unprecedented data that PRP derived from tumor-bearing animals is capable of accelerating wound closure and healing in a diabetic mouse model. The wound-healing parameters used to stage wound healing appeared markedly improved following a single topical application of TCPs when compared to both wild type derived PRP and spontaneously healing wounds.

[0086] In 1986, Dr. Harold Dvorak expressed the intriguing concept that "a tumor behaves as a wound that never heals". Although the original thought was conceptualized for tumor biology, impairments in the switching on and off of the angiogenic drive may partially explain delayed healing in chronic wounds as well. This study shows for the first time that an interaction between platelets and tumors ("tumor conditioning") leads to stimulation of platelet ability to make angiogenesis regulatory proteins more bioavailable. Tumor- conditioned platelets were found to stimulate all of the clinically relevant parameters of healing over both healthy PRP- and non-treated wounds. There was no significant quantitative difference in traditional stimulators of angiogenesis such as VEGF, bFGF or PDGF in the tumor conditioned PRP, yet, the proangiogenic response was qualitatively enhanced, suggesting a more orchestrated sequential presentation of these proteins. This is supported by the finding that healing, as measured by wound closure and angiogenesis, is induced only by intact platelets, and cannot be accomplished by delivering only the proteins, such as is the case with frozen platelets. Thus, while the angiogenic properties of frozen TPRP and PRP faded completely, frozen TPRP retained some proliferative potential compared to non-treated wounds and healthy PRP treatment.

[0087] The wound healing staging system developed in this study was based on the quantification of vasculature and cell proliferation in diabetic wounds⁵¹. The diabetic mouse, with its impaired angiogenesis, wound healing ^{35> 54}, and decreased production of growth factors ^{35> 55~58}, serves as a reproducible surrogate for the clinically relevant problem of recalcitrant wounds.

[0088] Platelets are the first responder to breaks in vascular integrity but their function is not limited to aggregation and coagulation at the site. It is now appreciated that there are different levels of platelet activation, and that upon adherence to the affected tissues, communicate freely through the open canallicular system. While not wishing to be bound by theory, this discovery that pro- and anti-angiogenic proteins are released in a sequential manner in response to PARl and PAR4 in intact platelets can explain why an unselective destruction, and a simultaneous release of positive and negative stimulators would have counterbalancing effects. Early in wound healing, angiogenesis is essential to initiate and support the repair process. New, small blood vessels form in the granulation tissue as a consequence of cell- and growth factor mediated events. Once healing has ceased, endothelial cells in the wound bed revert to a quiescent phenotype and start to undergo apoptosis in the early scar tissue, demonstrating that a timely switch between pro- and anti-angiogenesis is necessary to initiate and terminate the healing process, respectively⁶⁰. Such a process is evident herein in an animal model that resembles the phenotype of Type 2 diabetes in humans, wherein wound healing occurs in an accelerated fashion with PRP and T PRP treatments. It was observed that in the later stages of wound healing and by day 21, both PRP and T PRP were also able to modulate inhibition as wounds treated with these preparations demonstrated a down- regulation of angiogenesis while NT wounds showed increased levels of vessels.

[0089] A single topical dose of T PRP induced faster wound closure and increased cell proliferation of the wounded tissue compared to the other treatment groups, including wild type derived PRP. While not wishing to be bound by theory, altered platelet activation, orchestration and potential redistribution of the growth factors are a likely process that partly governs wound healing.

[0090] While not wishing to be bound by theory this is consistent with the concept that in non-malignant tissues, the sum of tissue-specific signals leads to the switching off of the pro- angiogenic signals in later stages of wound healing. One possible explanation is that the increase of thrombin and collagen in maturing wounds leads to activation of the receptor PAR4 and subsequent release of angiogenesis inhibitors from stromal cells and platelets. In contrast, the continuous growth of tumor tissues and the oncogene-facilitated stimulation of proangiogenic factors ⁶¹ prevents this switch in malignant tissues².

[0091] It is not believed that the effect observed is due to the plasma of the tumor-bearing animals. The common knowledge of platelet biology, as well as the half- lives of the reported growth factors in circulation generally favor the interpretation that the platelets, and not the plasma are responsible for the observed effect. For example, it is known that vascular endothelial growth factor exhibits a half-life in the order of seconds and is readily cleared from the circulation. Its existence and capacity to signal upon binding to its cognate receptor in vivo over an extended period of time - as suggested by these results - is essentially based on its heparin/heparan-sulphate binding properties. Additional support is provided by studies showing that platelets support the development and maturation of the endothelium and influence angiogenesis in vivo.¹⁹'²⁰

[0092] Considering the instability of the platelet-derived growth factors in the plasma, therefore, it is likely that the efficacy of employing autologous PRP in wound healing has been limited by the use of CaCl and thrombin in the preparation of a gel.²¹' ²² As a result of this conventional preparation ex vivo, platelets become activated and may release or redistribute their factors, thereby reducing their capacity to induce healing by appropriately delivering and presenting them at the site of the wound. While not wishing to be bound by theory, should the majority of the factors be released by the autologous platelet gels comprising activated platelets, this bolus-like dosing of growth factors may not be adequate to sustain wound healing over an extended period of time to due plasma clearance and proteolytic cleavage in vivo. Thrombin also causes significant fibrin cross-linking, considerably impairing cell access to the healing wound. Thus, while an activated platelet gel can be of use in compositions as described herein, a preferred aspect does not activate the platelets prior to application to a wound surface.

[0093] VEGF is one of the most studied angiogenic factors; in the blood stream it is transported mainly by platelets⁵ and its concentration in the serum may increase with tumor progression in many types of cancers^30"34. High doses of recombinant VEGF have been shown to have beneficial wound healing effects in animal models³⁵. In many instances attempts have been made to use VEGF as a prognostic factor³⁵'³⁶ but VEGF presence in platelets appears to have a more functional role. The inventors have demonstrated that platelets are able to present VEGF contained in its granules in a manner that enhances the pro-angiogenic effect of VEGF. In addition to VEGF-A and C, platelets have been documented to contain more than 14 pro-angiogenic growth factors (bFGF, HGF, angiopoietin-1, insulin-like growth factor- 1, epidermal growth factor, platelet derived growth factor), as well as inhibitors of angiogenesis, including platelet factor 4, thrombospondin-1, TGF-beta-1, plasminogen activator inhibitor type-1 (PAI-I), alpha2-antiplasmin and alpha2- macro globulin³⁷.

[0094] During wound healing angiogenesis is initially switched on and subsequently downregulated. This downregulation results in formation of poorly vascularized scar tissue, or may be a consequence of it. It appears that this temporal regulation of angiogenesis is well controlled under physiological conditions and healing and scar formation takes on average 10-14 days. Diabetes results in dysregulation of this process³⁸'³⁹ and healing is impaired. While some improvement of diabetic ulcer healing is achieved with angiogenic protein delivery³⁵, the use of platelet rich plasma is significantly more effective. Platelets of mice bearing tumors, which have been shown to contain significantly more angiogenesis regulators³⁰'⁴⁰'⁴¹ are even more efficacious in improving wound healing (Figure 1).

[0095] When compared to the administration of recombinant growth factors, platelets in wound healing can offer the advantage of serving as a drug delivery system.²³ The addition of angiogenic factors carried by platelets in tumor-bearing individuals to autologous platelets for wound healing preparations can be particularly advantageous.

Summary

[0096] The study of platelets spans over a century, but much of the acquired knowledge is focused on their behavior during clot formation. The contribution of platelets in the organization of extracellular matrix and the influence of this matrix on the formation of a neovasculature has been implied in many studies. ^47~49' ⁶² While not wishing to be bound by theory, the distinctive architecture of the extracellular matrix of TPRP treated wounds indicates a direct stimulation of platelets on stromal formation. In addition, there is evidence that the invasion of some skin tumors depends on stromal cell activation⁶³, which indicates an interplay exists between platelets and fibroblasts.

[0097] In summary, this study has established a novel biology based on the analogy between tumors and wounds. While diabetic wounds seem to lack some stimulation, tumors, standing on the opposite side of the angiogenic scale, seem to be excessively stimulated. Platelets interact with both environments, and the study of the details of these interactions and their functional consequences may lead to a better understanding of tumor and wound pathophysiology and improvements in therapeutic options.

[0098] All references noted herein are hereby incorporated herein in their entirety, including figures and tables.

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Claims

1. A composition for promoting wound healing, the composition comprising platelets comprising an exogenous angiogenesis promoting factor and a pharmaceutically acceptable carrier.

2. The composition of claim 1 wherein said platelets are autologous to said individual.

3. The composition of claim 1 or 2 wherein said angiogenesis regulating factor is selected from the group consisting of agents that activate the VEGF pathway, agents that activate the neuropilin 1 & 2 pathways, VEGF-A and C, bFGF, HGF, angiopoietin-1, insulin-like growth factor- 1, epidermal growth factor, platelet derived growth factor, platelet factor 4, thrombospondin-1, TGF-beta-1, plasminogen activator inhibitor type-1 (PAI-I, alpha2-antiplasmin and alpha2- macroglobulinVEGFRl (flt-1), VEGFR2 (flk-2), VEGFR3 (flt-4), heparin sulfate proteoglycan, VEGF121, VEGF145, VEGF165, VEGF168, VEGF189, VEGF -B and -D, PLGF 1, PLGF2, HIV-I TAT, Sema-E, Sema-III, Sema-IV, bFGF, PDGFR, EGFR, and IGFR.

4. Use of a composition comprising platelets comprising an exogenous angiogenesis regulating factor and a pharmaceutically acceptable carrier for promotion of wound healing.

5. The use of claim 4 wherein said platelets are autologous to said individual.

6. The use of claim 4 or 5 wherein said angiogenesis regulating factor is selected from the group consisting of agents that activate the VEGF pathway, agents that activate the neuropilin 1 & 2 pathways, VEGF-A and C, bFGF, HGF, angiopoietin-1, insulin-like growth factor- 1, epidermal growth factor, platelet derived growth factor, platelet factor 4, thrombospondin-1, TGF-beta-1, plasminogen activator inhibitor type-1 (PAI-I, alpha2-antiplasmin and alpha2- macroglobulinVEGFRl (flt-1), VEGFR2 (flk-2), VEGFR3 (flt-4), heparin sulfate proteoglycan, VEGF121, VEGF145, VEGF165, VEGF168, VEGF189, VEGF -B and -D, PLGF 1, PLGF2, HIV-I TAT, Sema-E, Sema-III, Sema-IV, bFGF, PDGFR, EGFR, and IGFR.

7. The use of claim 4, 5, or 6 or any one of the preceding claims wherein said wound is selected from a burn, an incision, a laceration, an abrasion, an ulcer, a diabetic wound, a venous stasis wound, a vascular wound, a radiation wound, a steroid wound or a bony defect.

8. The use of any one of claims 4-7, or any one of the preceding claims wherein said wound is a diabetic ulcer.

9. The use of any one of claims 4-7 or any one of the preceding claims wherein said wound is a decubitus ulcer.

10. The use of any one of claims 4-9 or any one of the preceding claims, further comprising the step of loading said platelets with said exogenous angiogenesis regulating factor.

11. The use of claim 10 or any one of the preceding claims wherein said loading comprises contacting platelets with said factor.

12. The use of any one of claims 4-11 or any one of the preceding claims wherein said platelets comprise at least two angiogenesis promoting factors.

13. The use of any one of claims 4-12 or any one of the preceding claims wherein said composition comprises a platelet gel comprising said platelets and said angiogenesis regulating factor.

14. The use of claim 13 wherein said gel is formed by contacting said platelets with calcium and a platelet activating agent.

15. The use of claim 14 wherein said platelet activating agent is selected from the group consisting of thrombin, collagen, serotonin, ADP, acetylcholine and combinations thereof.

16. The use of any one of claims 4-15, or any one of the preceding claims wherein said angiogenesis regulating factor comprises a PAR-I agonist.

17. The use of claim 16 wherein said PAR-I agonist is selected from the group consisting Of TFLLR-NH₂ , TFLLR NPNDK-NH₂, SFLLRNPNDKYEPF-NH₂, and SFLLRN-NH₂.

18. The use of any one of claims 4-17 or any one of the preceding claims wherein said angiogenesis promoting factor comprises a PAR-4 antagonist.

19. The use of claim 18 wherein said PAR-4 antagonist is selected from the group consisting of transcinnamoyl- YPGKF-NH₂, and ethyl 4-(l-benzyl-lH-indazol-3- yl)benzoate.

20. The use of any one of claims 4-19 or any one of the preceding claims wherein said platelets further comprise an exogenous angiogenesis inhibiting factor.

21. Use of a platelet composition comprising an exogenous angiogenesis regulating factor in the preparation of a medicament for the promotion of wound healing.

22. The use of claim 21 wherein said wound is selected from a burn, an incision, a laceration, an abrasion, an ulcer, a diabetic wound, a venous stasis wound, a vascular wound, a radiation wound, a steroid wound or a bony defect.

23. The use of claim 21 or 22 wherein said wound is a diabetic ulcer.

24. The use of claim 21 or 22 wherein said wound is a decubitus ulcer.

25. The use of any one of claims 21-24 or any one of the preceding claims, further comprising the step of loading said platelets with said exogenous angiogenesis regulating factor.

26. The use of claim 25 wherein said loading comprises contacting platelets with said factor.

27. The use of any one of claims 21-26 or any one of the preceding claims wherein said platelets comprise at least two angiogenesis promoting factors.

28. The use of any one of claims 21-28 or any one of the preceding claims wherein said composition comprises a platelet gel comprising said platelets and said angiogenesis regulating factor.

29. The use of claim 28 wherein said gel is formed by contacting said platelets with calcium and a platelet activating agent.

30. The use of claim 29 wherein said platelet activating agent is selected from the group consisting of thrombin, collagen, serotonin, ADP, acetylcholine and combinations thereof.

31. The use of any one of claims 21-30 or any one of the preceding claims wherein said angiogenesis regulating factor comprises a PAR-I agonist.

32. The use of claim 31 wherein said PAR-I agonist is selected from the group consisting Of TFLLR-NH₂ , TFLLRNPNDK-NH₂, SFLLRNPNDKYEPF-NIL, and SFLLRN-NH₂.

33. The use of any one of claims 21-32 or any one of the preceding claims wherein said angiogenesis promoting factor comprises a PAR-4 antagonist.

34. The use of claim 33 wherein said PAR-4 antagonist is selected from the group consisting of transcinnamoyl- YPGKF-NH₂, and ethyl 4-(l-benzyl-lH-indazol-3- yl)benzoate.

35. The use of any one of claims 21-34 or any one of the preceding claims wherein said platelets further comprise an exogenous angiogenesis inhibiting factor.

36. A method of promoting wound healing in an individual in need thereof, the method comprising the step of contacting a wound with a composition comprising isolated platelets comprising an exogenous angiogenesis regulating factor, wherein said contacting promotes healing of said wound.

37. The method of claim 36 wherein said platelets are autologous to said individual.

38. The method of claim 36 or 37 wherein said angiogenesis regulating factor is selected from the group consisting of agents that activate the VEGF pathway, agents that activate the neuropilin 1 & 2 pathways, VEGF-A and C, bFGF, HGF, angiopoietin-1, insulin-like growth factor- 1, epidermal growth factor, platelet derived growth factor, platelet factor 4, thrombospondin-1, TGF-beta-1, plasminogen activator inhibitor type-1 (PAI-I, alpha2-antiplasmin and alpha2- macroglobulinVEGFRl (flt-1), VEGFR2 (flk-2), VEGFR3 (flt-4), heparin sulfate proteoglycan, VEGF121, VEGF145, VEGF165, VEGF168, VEGF189, VEGF -B and -D, PLGF 1, PLGF2, HIV-I TAT, Sema-E, Sema-III, Sema-IV, bFGF, PDGFR, EGFR, and IGFR.

39. The method of claim 36, 37, 38, or any one of the preceding claims wherein said wound is selected from a burn, an incision, a laceration, an abrasion, an ulcer, a diabetic wound, a venous stasis wound, a vascular wound, a radiation wound, a steroid wound or a bony defect.

40. The method of any one of claims 36-39 or any one of the preceding claims wherein said wound is a diabetic ulcer.

41. The method of any one of claims 36-39 or any one of the preceding claims wherein said wound is a decubitus ulcer.

42. The method of any one of claims 36-41 or any one of the preceding claims, further comprising the step of loading said platelets with said exogenous angiogenesis regulating factor.

43. The method of claim 42 wherein said loading comprises contacting platelets with said factor.

44. The method of any one of claims 36-43 or any one of the preceding claims wherein said platelets comprise at least two angiogenesis promoting factors.

45. The method of any one of claims 36-44 or any one of the preceding claims wherein said composition comprises a platelet gel comprising said platelets and said angiogenesis regulating factor.

46. The method of claim 45 wherein said gel is formed by contacting said platelets with calcium and a platelet activating agent.

47. The method of claim 46 wherein said platelet activating agent is selected from the group consisting of thrombin, collagen, serotonin, ADP, acetylcholine and combinations thereof.

48. The method of any one of claims 36-47 or any one of the preceding claims wherein said angiogenesis regulating factor comprises a PAR-I agonist.

49. The method of claim 48 wherein said PAR-I agonist is selected from the group consisting Of TFLLR-NH₂ , ITLLRNPNDK-NH;, SFLLRNPNDKYEPF-NH₂, and SFLLRN-NH₂.

50. The method of any one of claims 36-49 or any one of the preceding claims wherein said angiogenesis promoting factor comprises a PAR-4 antagonist.

51. The method of claim 50 wherein said PAR-4 antagonist is selected from the group consisting of transcinnamoyl- YPGKF-NH₂, and ethyl 4-(l-benzyl-lH-indazol-3- yl)benzoate.

52. The method of any one of claims 36-51 or any one of the preceding claims wherein said platelets further comprise an exogenous angiogenesis inhibiting factor.

53. A method of delivering angiogenesis regulators to a wound site, the method comprising contacting said wound site with a composition comprising isolated platelets comprising an exogenous angiogenesis regulating factor, whereby said angiogenesis regulating factor is delivered to said wound site.

54. The method of claim 53 wherein said platelets are autologous to said individual.

55. The method of claim 53 or 54 wherein said angiogenesis regulating factor is selected from the group consisting of agents that activate the VEGF pathway, agents that activate the neuropilin 1 & 2 pathways, VEGF-A and C, bFGF, HGF, angiopoietin-1, insulin-like growth factor- 1, epidermal growth factor, platelet derived growth factor, platelet factor 4, thrombospondin-1, TGF-beta-1, plasminogen activator inhibitor type-1 (PAI-I, alpha2-antiplasmin and alpha2- macroglobulinVEGFRl (flt-1), VEGFR2 (flk-2), VEGFR3 (flt-4), heparin sulfate proteoglycan, VEGF121, VEGF145, VEGF165, VEGF168, VEGF189, VEGF -B and -D, PLGF 1, PLGF2, HIV-I TAT, Sema-E, Sema-III, Sema-IV, bFGF, PDGFR, EGFR, and IGFR.

56. The method of any one of claims 53-56 or any one of the preceding claims further comprising contacting said platelets that have been contacted with an angiogenesis regulating factor with a platelet activating agent.

57. The method of claim 56 wherein said platelet activating agent is selected from the group consisting of thrombin, collagen, serotonin, ADP, acetylcholine and combinations thereof.

58. A method of preparing a wound healing composition, the method comprising contacting platelets with an exogenous angiogenesis regulating factor and a pharmaceutically acceptable carrier.

59. The method of claim 58 wherein said angiogenesis regulating factor is selected from the group consisting of agents that activate the VEGF pathway, agents that activate the neuropilin 1 & 2 pathways, VEGF-A and C, bFGF, HGF, angiopoietin-1, insulin-like growth factor- 1, epidermal growth factor, platelet derived growth factor, platelet factor 4, thrombospondin-1, TGF-beta-1, plasminogen activator inhibitor type-1 (PAI-I, alpha2-antiplasmin and alpha2- macroglobulinVEGFRl (flt-1), VEGFR2 (flk-2), VEGFR3 (flt-4), heparin sulfate proteoglycan, VEGF121, VEGF145, VEGF165, VEGF168, VEGF189, VEGF -B and -D, PLGF 1, PLGF2, HIV-I TAT, Sema-E, Sema-III, Sema-IV, bFGF, PDGFR, EGFR, and IGFR.

60. The method of claim 58 or 59 further comprising contacting said platelets that have been contacted with an angiogenesis regulating factor with a platelet activating agent.

61. The method of claim 60 wherein said platelet activating agent is selected from the group consisting of thrombin, collagen, serotonin, ADP, acetylcholine and combinations thereof.

62. A kit for the preparation of a wound healing composition, the kit comprising an angiogenesis regulating factor and a platelet preparation.

63. A kit for the preparation of a wound healing composition, the kit comprising an angiogenesis regulating factor and a container for holding and/or incubating platelets.