1. EFFECT OF ZEOLITE (QUIKCLOT TM ) IN REDUCING BLOOD LOSS IN RATS AND THE MECHANISM OF ACTION OF ZEOLITE ON BLOOD IN VITRO RAMON BONEGIO, ROBERT FUHRO, RITHY SREY, GINA RAGNO, AND C. ROBERT VALERI NAVAL BLOOD RESEARCH LABORATORY, BOSTON UNIVERSITY SCHOOL OF MEDICINE, BOSTON, MA This work was supported by the U.S. Navy (Office of Naval Research Contract N00014-02-1-0939). The opinions or assertions contained herein are those of the authors and are not to be construed as official or reflecting the views of the Navy Department or Naval Service at large.

2. BACKGROUND The restoration of hemostasis at surgical bleeding sites is an important approach to reduce the need for blood products which are associated with significant risks. Safe and therapeutically effective hemostatic agents are needed and several potential substances are being investigated for safety and therapeutic benefit. This study was done to evaluate the safety and therapeutic effectiveness of zeolite (QuikClot™) to restore hemostasis in the rodent and to assess the mechanism of action of zeolite on blood in vitro.

3. STUDY DESIGN AND METHODS In Vitro Studies: In vitro studies were done to assess the effect of 1 gram of zeolite with and without CaCl 2 added to 10 ml of platelet rich plasma on the thromboelastogram measurement. The release of platelet-derived growth factor AA, transforming growth factor beta (TGF-b) and osteocalcin from platelets treated with zeolite and calcium was measured. Platelet-rich plasma treated with zeolite without calcium was then assayed for its ability to stimulate fibroblast proliferation and survival in cell culture studies using an embryonic mouse fibroblasts cell line (3T3-Swiss Albino). In Vivo Studies: Studies were performed in rats subjected to anesthesia and puncture of the abdominal aorta using a 23 gauge needle. Zeolite 2g was placed intra abdominally before pressure was applied to the puncture site for 30 seconds. This was compared to pressure alone(control). These studies were done to determine if the QuikClot™ method reduced blood loss and mortality in these rodents. Survival was assessed over a 30- minute period and then the rodents were euthanized. Additional studies were done where survival was assessed after 2 weeks.

4. RESULTS In the in vitro studies, the addition of zeolite with and without CaCl 2 to human platelet rich plasma decreased the R time of the thromboelastogram test which indicated activation of the platelets (Table 1). The zeolite reacted with the human platelet-rich plasma to quickly form a clot with the conversion of fibrinogen to fibrin. The addition of zeolite without CaCl 2 to platelet-rich plasma resulted in activation of the platelets with the release of significant amounts of platelet-derived growth factor AA (PDGF AA) and transforming growth factor beta 1 compared to that of untreated platelet-rich plasma. Osteocalcin was not released from the platelets following stimulation with zeolite without CaCl 2. Zeolite without CaCl 2 treated platelet-rich plasma produced a platelet lysate that stimulated fibroblast proliferation in the tissue culture of fibroblasts isolated from renal cells (Figure 1). The in vivo studies demonstrated that in 22 rats the zeolite and pressure treatment resulted in significantly reduced blood loss compared to the pressure alone (3.6 + 2.6 ml, n=12 compared to 8.6 + 2.0 ml, n=10, Figure 2). Ninety five percent of the rodents treated with zeolite survived the 30 minute period whereas 50% of those treated with the pressue alone did not survive. Twenty zeolite treated rats and five control rats that survived the initial aortic puncture were followed for 14 days. While all the control animals survived without complications, 48% of the animals exposed to intra-abdominal zeolite died during the two weeks following aortic puncture. Surviving zeolite treated animals commonly developed splenomegaly or intra-abdominal adhesions (Figure 2).

5. CONCLUSION The in vitro testing demonstrated that zeolite without CaCl2 activated stored platelets to release platelet derived growth factors (PDGF AA) and transforming growth factor beta 1. This stimulated fibroblast proliferation in cell culture. We saw that zeolite improves the early survival of rats subjected to severe surgical bleeding by the reduction in blood loss. The long-term safety of the zeolite when placed into the abdominal cavity is however a concern as many of the treated animals developed intra- abdominal complications. These complications are currently under investigation.

6. TABLE 1 TREATMENT OF PLATELET RICH PLASMA WITH ZEOLITE WITH AND WITHOUT CALCIUM SAMPLE TREATMENTSAMPLE IDR TIME (min)K TIME (min)ANGLE (deg)MA (mm)G (Kd/sec)CI PRP WITH 0.2 M CALCIUM 110.91.078.470.211.82.0 PRP WITH 0.2 M CALCIUM 219.81.769.8 11.2-0.1 PRP WITH 0.2 M CALCIUM 312.31.475.477.717.43.0 PRP WITH 0.2 M CALCIUM 420.81.772.077.116.90.9 MEAN16.01.573.973.714.31.5 SD5.10.33.84.33.31.3 ` PRP/ZEOLITE56.32.854.373.313.7 PRP/ZEOLITE67.93.651.667.610.43.0 PRP/ZEOLITE76.80.879.785.629.85.6 MEAN7.02.461.975.518.04.3 SD0.81.415.59.210.41.8 PRP/ZEOLITE/CALCIUM80.20.883.868.210.74.2 PRP/ZEOLITE/CALCIUM90.10.883.267.110.24.1 PRP/ZEOLITE/CALCIUM100.8 85.482.824.16.5 PRP/ZEOLITE/CALCIUM111.50.884.181.522.06.1 MEAN0.70.884.174.916.85.2 SD0.60.00.98.47.31.3 CODE: R = time of detectable clot with minimum fibrin deposit (2mm in amplitude) K=time to reach minimum clot strength of 20 mm in amplitude ANGLE =the angle of the clot size MA=maximum clot strength G=clot strength in Kd/sec CI=clotting index where normal is between -3 and 3; greater than 3 = hypercoagulability

7. FIGURE 1A: INCUBATION OF FIBROBLASTS (3T3) WITH PLATELET LYSATE PREPARED FROM PLATELET-RICH PLASMA TREATED WITH ZEOLITE DMEM+LG = Dulbecco’s modified enriched medium + low glucose P = platelet lysate studied at dilutions of 1:10, 1:50, 1:100, 1:250 and 1:1000 Fibroblast (3T3) proliferation response to platelet lysate prepared from platelet rich plasma treated with zeolite 0 1000 2000 3000 4000 5000 6000 7000 DMEM+LG P1:10P1:50 P1:100P1:250 P1:1000 CPM (H3-Thymidine) 3T3 Thymidine uptake MEAN+SEM,n=3

8. Figure 1b:The incubation of human dermal fibroblasts (Coriell, AG1512) with platelet lysate prepared from platelet-rich plasma stored at 22 C for 2 to 21 days and then treated with zeolite Platelets stored at 22 C for 2 days, 3 days, 5 days and 21 days (A) Fibroblast proliferation after stimulation with the platelet lysate (B) Total protein concentration (mg/ml) (C) Concentration of PDGF-AA in pg/ml. (D) Concentration of TGF-  in pg/ml.

9. Figure a: Days at 22C: 2 3 5 21 FIBROBLAST PROLIFERATION INDEX Fibroblast proliferation index

10. Figure b: Days at 22C: 2 3 5 21 TOTAL PROTEIN CONCENTRATION Total protein concentration (mg/ml)

11. Days at 22C: Figure c: 2 3 5 21 PLATELET DERIVED GROWTH FACTOR AA Platelet derived growth factor AA (pg/ml)

12. Figure d: Days at 22C: 2 3 5 21 TRANSFORMING GROWTH FACTOR  1 Transforming growth factor  1 (pg/ml)

13. Figure 2a: Survival curve of rats subjected to a standardized abdominal aortic puncture and treated with 2g of zeolite (QuikClot™) and pressure compared to pressure alone.

14. Figure 2b: Outcome of zeolite treated rats over the 14 days following aortic puncture. Forty eight percent of the zeolite treated animals that survived the initial aortic puncture procedure developed intra-abdominal complication related to the initial injury or the zeolite treatment. Although 50% of the control animals died initially, none of the control rats that survived initial injury developed abdominal complications. Zeolite-treated rats 1 rat died <1 hour of hemorrhage 1 rat died at 2 days - post mortem lung problems (redness) 1 rat euthanased at 4 days - Rear leg paralysis, blood at nostrils, possible peritonitis 3 rats died at 4 days (no post-mortem) 1 rat died at 10 days (no post-mortem) 1 rat died at 11 days (no post-mortem) 1 rat died at 12 days (no post-mortem) 1 rat died at 13 days (no post-mortem) 10 rats were sacrificed at 14 days 1 Rat: no abnormalities noted 5 Rats: enlarged spleen 1 Rat:Rear leg paralysis, enlarged spleen and liver, hydronephrotic kidney 1 Rat:Bowel adhesions, abnormal kidneys 1 Rat:Enlarged spleen, bowel adhesions 1 Rat:Obstructed bowel due to adhesions Control 5 rats died within 1 hour 5 rats euthanased at 14 days with no abnormal findings