Procoagulant peptides and use thereof

[0038] Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying figures.

[0039] With specific reference now to the figures in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description together with the drawings makes apparent to those skilled in the art, how embodiments of the invention may be practiced. Dimensions of components and features shown in the figures are generally chosen for convenience and clarity of presentation and are not necessarily shown to scale. The figures are listed below.

[0040] FIGS. 1A-1C include vertical bar graphs and thromboelastography (TEG) curves, showing that peptide 16 and peptide 16AC increase coagulation. Peptide 16 and 16AC were studied in an assay of purified proteins that included lipidated TF (4 nM), factor VIIa (40 nM), factor X (1.4 μM) and assay buffer. Factor X activated form (Xa) level was studied using a chromogenic substrate (1 mM). The peptides at a dose of 5 μg/ml and 10 μg/ml (3.75 μM and 7.5 μM, respectively) significantly increased activation of factor X compared to control (1A), thus increasing activation of the coagulation system. Bovine factor Xa diluted in the assay buffer was used to generate a standard curve. Without addition of TF (two right columns), no factor Xa was generated. These results represent a mean of triplicate and range. *p<0.01, **p<0.001, ***p<0.0001. (1B) A citrated pooled normal human blood sample (n=3) was left for 4 hours at room temperature in order to expose TF in bloods cells. Following this period, the peptides 16 and 16AC were added (5 μg/ml) along with calcium. The peptides significantly reduced the time to clot formation (R) and increased thrombus strength, as indicated by a greater maximal amplitude (MA). Representative TEG curves are shown. (1C) The study presented in FIG. 1B was repeated 3 times with different pooled plasma. These results represent a mean of triplicate and range. *p<0.01, **p<0.001. The modified peptide 16AC demonstrated significantly better effect on clot formation compared to peptide 16. Peptide 16 (non-modified); peptide 16 comprising a C-terminal amid group (16A); peptide 16 comprising an N-terminal acetyl group (16C); and peptide 16 comprising a C-terminal amid group and an N-terminal acetyl group (16AC). In 1B: for control—SP min=11.2; R min=12.8; K min=3.8, Angle deg=52.0; and MA mm=47.9, for Peptide 16—SP min=5.2; R min=6.4; K min=2.8, Angle deg=63.3; and MA mm=50.4, and for Peptide 16AC—SP min=4.7; R min=5.6; K min=2.5, Angle deg=63.1; and MA mm=53.8.

[0041] FIGS. 2A-2I include vertical bar graphs showing the effect of modified heparanase procoagulant peptides of the invention using different rat bleeding models. Two rat models of uncontrolled hemorrhage were applied. The models include spleen transection (2A) and liver laceration (2B). Blood loss was recorded every 2 minutes for the first 10 minutes after injury and at 5-minutes intervals thereafter for a total of 30 minutes. Shed blood were collected by placing reweighed gauze below the organ targeted for injury. The gauze was changed and weighed at the blood-loss intervals. Peptides 16 and 16AC were injected subcutaneously (150 μg/kg) half an hour before surgery (2A-2B) or intravenously (I.V.) in the saphenous vein (150 μg/kg) 2 minutes following the trauma (2C, 2D, 2H, and 2I). The third route of administration was topical application (150 μg/kg) on the incision site (2E-2F). The peptides were dissolved in phosphate-buffered saline (PBS) and added at a rate of a drop every 5 seconds. Control group (‘C’) was administered (either subcutaneously, intravenously, or topically) with the vehicle phosphate buffered saline (PBS). A significant reduction in bleeding (2A-2F) and in bleeding time (2G) was observed when the peptides were applied. The difference in the rat weight before and at the end of the procedure and the pads with blood weight were significantly decreased in the study group compared to control. The modified peptide 16AC demonstrated a significant better effect to reduce the bleeding compared to peptide 16. n—number of mice. Mann-Whitney U test was used. Results represent mean±SD. *p<0.01, **p<0.001. In addition to Peptide 16 and Peptide 16AC, Peptide 16C, and Peptide 16A were also analyzed and compared in (2H and 2I).

[0042] FIGS. 3A-3D include vertical bar graphs showing that modified heparanase procoagulant peptides of the invention decrease bleeding in hemophilic mice. Hemophilic mice (VIII-KO mice; C57BL/6 background, strain name B6;129S4-F8tm1Kaz/J) were studied. (3A) Peptides 16 or 16AC were injected subcutaneously at the flank in doses 150, 225, 300, 375 μg/kg. A mouse-tail bleeding time model was performed. The peptides were dissolved in phosphate-buffered saline (PBS). Control (0) group was injected with the vehicle phosphate buffered saline (PBS). (3D) is identical to (3A) with the exception that peptide 16 C (comprising a C-terminal amid group) and peptide 16A (comprising an N-terminal acetyl group) were also administered in the disclosed doses. In a similar study, the peptides were topically applied on the tail immediately following the incision of the tail at a rate of a drop every 5 seconds (3B). Comparable results were obtained when peptides were dissolved in white soft paraffin (Rekah Pharma Industry, Israel) and applied in a very thin layer (0.25 μm) on the flanked, following hair removal, 1 hour prior to tail incision (3C). A significant decrease in bleeding time was observed in the hemophilic mice in a dose dependent manner. The modified peptide 16AC demonstrated a significant better effect to reduce the bleeding time compared to peptide 16. Mann-Whitney U test was used. Results represent mean±SD. *p<0.05.

[0043] FIGS. 4A-4E include images, micrographs, and graphs showing that that modified heparanase procoagulant peptides of the invention enhance wound healing. (4A) Full thickness incision of 10 mm was made in the back skin of ICR (normal, no genetic background) mice. On alternate days, for one week after surgery, peptide 16 or peptide 16AC was injected subcutaneously (S.C.) opposite to the wound at the indicated dose. Control group was injected the vehicle (PBS). Modified peptide 16AC demonstrated significantly better effect on wound size healing (4A; black arrows, and 4B). Similar results were observed when the peptides were topically applied as a cream preparation (4C). n—number of mice. Mann-Whitney U test was used. Results represent mean±SD, **p<0.001, ***p<0.0001. (4D) At the end of the experiment the mice were sacrificed and the skin tissue from the wound site excised, and histologically analyzed using hematoxylin and eosin (H&E) staining. Representative images are shown. A significant increase of capillaries (4D; black arrows) was observed in the skin tissue of the treatment groups, compared to control (4E). The number of capillaries presented reflects the median and range of five different fields. Mann-Whitney U test was used. Results represent mean±SD, *p<0.01, **p<0.001.

[0044] FIGS. 5A-5B include vertical bar graphs showing that heparanase procoagulant peptides induce human umbilical vein endothelial cells (HUVEC) proliferation. (5A) HUVEC cells were seeded in 96 wells plate (5,000 cells/well). Increasing concentration of peptides 16 and 16AC (2.5 μg/ml, and 5 μg/ml) were added to the cells. After 48 hour, using the XTT assay, a significant increase in cell proliferation was observed (*P<0.05). (5B) Similar experiment as described in 5A, but 10 ng/ml of peptide 7 (TFPI-2 derived inhibitory peptide) was added to the cells for 1 hour prior to the addition of peptide 16 (5 μg/ml) or 16AC (5 μg/ml). Peptide 7 significantly inhibited the proliferative effect of any one of peptides 16 and 16AC.

[0045] FIGS. 6A-6F include micrographs of immunohistochemistry assays showing that heparanase procoagulant peptides increase the level of heparanase in HUVECs. Ten (10) μg/ml of peptide 16 (6B-6C) or 10 μg/ml of peptide 16AC (6E-6F) was added to HUVEC cells and compared to control cells (6A-6D). After 30 min or overnight incubation at 37° C., the cells were fixed using formaldehyde 4%, permeabilized, blocked and reacted with specific heparanase antibody. A strong staining after overnight incubation in the treated cells compared to the control, indicates heparanase over-expression in treated cells. Images were visualized through ×50 magnitude, with 0.82 MDC objective lens, captured with a Nikon E995 digital camera (Nikon, Tokyo, Japan), and processed with Adobe Photoshop software (Adobe Systems, San Jose, CA, USA).

[0046] FIGS. 7A-7F include micrographs of immunohistochemistry assays showing that heparanase procoagulant peptides increase the level of TF in HUVECs. Ten (10) μg/ml of peptide 16 (7B-7C) or 10 μg/ml of peptide 16AC (7E-7F) was added to HUVEC cells and compared to control cells (7A-7D). After 30 min or overnight incubation at 37° C., the cells were fixed using formaldehyde 4%, permeabilized, blocked and reacted with specific TF antibody. Images were visualized through ×50 magnitude, with 0.82 MDC objective lens, captured with a Nikon E995 digital camera (Nikon, Tokyo, Japan), and processed with Adobe Photoshop software (Adobe Systems, San Jose, CA, USA).

[0047] FIGS. 8A-8F include micrographs of immunohistochemistry assays showing that heparanase procoagulant peptides increase the level of TFPI in HUVECs. Ten (10) μg/ml of peptide 16 (8B-8C) or 10 μg/ml of peptide 16AC (8E-8F) was added to HUVEC cells and compared to control cells (8A-8D). After 30 min or overnight incubation at 37° C., the cells were fixed using formaldehyde 4%, permeabilized, blocked and reacted with specific TFPI-1 antibody. Images were visualized through ×50 magnitude, with 0.82 MDC objective lens, captured with a Nikon E995 digital camera (Nikon, Tokyo, Japan), and processed with Adobe Photoshop software (Adobe Systems, San Jose, CA, USA).

[0048] FIGS. 9A-9F include micrographs of immunohistochemistry assays showing that heparanase procoagulant peptides increase the level of TFPI-2 in HUVECs. Ten (10) μg/ml of peptide 16 (9B-9C) or 10 μg/ml of peptide 16AC (9E-9F) was added to HUVEC cells and compared to control cells (9A-9D). After 30 min or overnight incubation at 37° C., the cells were fixed using formaldehyde 4%, permeabilized, blocked and reacted with specific TFPI-2 antibody. Images were visualized through ×50 magnitude, with 0.82 MDC objective lens, captured with a Nikon E995 digital camera (Nikon, Tokyo, Japan), and processed with Adobe Photoshop software (Adobe Systems, San Jose, CA, USA).

[0049] FIGS. 10A-10D include micrographs of immunohistochemistry assays showing that heparanase procoagulant peptides increase the level of heparanase, TF, TFPI and TFPI-2. Full thickness incision was made in the back skin of ICR mice. Each group included 5 mice. From the day of surgery either peptide 16 or 16AC was injected subcutaneous opposite to the wound, every other day for a week. At the end of the experiment the mice were sacrificed and the wound skin section was analyzed by immunostaining. A significant increase in the expression of heparanase (10A), TF (10B), TFPI-1 (10C), and TFPI-2 (10D) were observed in the micro-vessels (endothelial cell and intraluminal). Images were visualized through ×50 magnitude, with 0.82 MDC objective lens, captured with a Nikon E995 digital camera (Nikon, Tokyo, Japan), and processed with Adobe Photoshop software (Adobe Systems, San Jose, CA, USA).

[0050] FIG. 11 includes micrographs showing that procoagulant peptides increase endothelial cells migration. HUVEC cells were seeded in 35 mm dishes. At confluence of 90%, artificial gap was created using 1,000-μl standard pipette tip. Plate was washed twice with PBS to remove cell debris and incubated with either peptide 16 or 16AC (10 μg/ml). Representative photos of the scratched area after 4 hours are presented. Images were visualized through ×50 magnitude, with 0.82 MDC objective lens, captured with a Nikon E995 digital camera (Nikon, Tokyo, Japan), and processed with Adobe Photoshop software (Adobe Systems, San Jose, CA, USA).

[0051] FIG. 12 includes a vertical bar graph and a micrograph of a western blot analysis. Procoagulant peptide 16AC was added for 2 hours to transfected HEK-293 cells with full length TF (TF) or truncated TF devoid of the intracellular part (ΔTF). When the TF was devoid of the intracellular part, an increase in p-p38 intracellular signaling was not observed, thus indicating that TF is a receptor to heparanase at the procoagulant domain. Control—vector only (Vo); transfection with the empty plasmid, with no additional DNA.