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In Vitro Effects of Poly-N-Acetyl Glucosamine on the Activation of Platelets in Platelet-Rich Plasma with and without Red Blood Cells

Valeri, C Robert MD; Srey, Rithy MS, CCP; Tilahun, Daniel MS, CCP; Ragno, Gina BA

Journal of Trauma-Injury Infection & Critical Care: July 2004 - Volume 57 - Issue 1 - pp S22-S25
Article Titles

Background: This study was performed to assess the effect of poly-N-acetyl glucosamine fiber slurry on plasma clotting proteins, platelets, and red blood cells in the clotting of the blood.

Methods: Citrate phosphate dextrose whole blood was stored at 22°C for 48 hours to prepare platelet-poor plasma, platelet-rich plasma (PRP), and PRP plus red blood cells with hematocrit values of 20%, 35%, and 45% with and without an equal volume of poly-N-acetyl glucosamine fibers (1 mg/mL 0.9% NaCl).

Results: Thromboelastogram data show that poly-N-acetyl glucosamine fibers (p-GlcNAc) significantly reduced the R time in platelet-poor plasma, PRP, and PRP supplemented with red blood cells. Poly-N-acetyl glucosamine fibers increased, but not significantly, Annexin V and factor X binding to platelets, platelet microparticles, and red blood cell Annexin V binding. Poly-N-acetyl glucosamine fibers increased the production of thromboxane B2 by PRP.

Conclusion: Poly-N-acetyl glucosamine slurry activates platelets.

From the Naval Blood Research Laboratory, Boston University School of Medicine, Boston, Massachusetts.

Submitted for publication January 28, 2004.

Accepted for publication February 20, 2004.

Supported by U.S. Navy Office of Naval Research contract N00014-00-1-055.

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 the Naval Service at large.

Presented, in part, at the Harvard University Department of Surgery Symposium, Clinical Activity and Mode of Action of the Hemostatic Agent Poly-N-Acetyl Glucosamine, February 25, 2003, Boston, Massachusetts.

Address for reprints: C. Robert Valeri, MD, Naval Blood Research Laboratory, Boston University School of Medicine, 615 Albany Street, Boston, MA 02118; email: navblood@bu.edu.

The poly-N-acetyl glucosamine fiber-based SyvekPatch used in conjunction with local pressure has been shown to reduce bleeding at sites of blood loss. The mechanism by which the poly-N-acetyl glucosamine (p-GlcNAc) fiber patch acts may be related to local vasoconstriction and to the activation of the clotting system of blood. This study was performed to assess the effect of p-GlcNAc fiber slurry on plasma clotting proteins, platelets, and red blood cells.

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PATIENTS AND METHODS

All blood donors who participated in this study met the requirements of the American Association of Blood Banks. None of the participants had taken any medications for 10 days before donating blood.

Four hundred fifty milliliters of whole blood was collected into 63 mL of citrate phosphate dextrose anticoagulant and stored at room temperature during transportation from the Oklahoma Blood Institute, Oklahoma City, Oklahoma. After storage at room temperature (22°C) for 48 hours, the unit of whole blood was centrifuged at 1,615 × g for 4 minutes to prepare the platelet-rich plasma (PRP). The PRP was concentrated by centrifugation at 4,500 × g for 5 minutes to prepare platelet-poor plasma (PPP). The autologous red blood cells were added to the PRP to achieve hematocrit values of 20%, 35%, and 45%.

PPP, PRP, and PRP plus red blood cells with hematocrit values of 20%, 35%, and 45% were treated with or without 0.2 mol/L CaCl2 alone or with an equal volume of poly-N-acetyl glucosamine fiber slurry at 1.0 mg/1.0 mL of 0.9% NaCl (Marine Polymer Technologies, Inc., Danvers, MA) or a combination of an equal volume of 0.2 mol/L CaCl2 and poly-N-acetyl glucosamine fiber slurry (1 mg/1 mL of 0.9% NaCl). Blood samples were obtained before and after storage at 37°C for 30 minutes to assay thromboxane B2, prothrombin fragment 1.2, and D-dimer by the enzyme-linked immunoassays with specific monoclonal antibodies1 and fibrinogen by a clotting assay.2 We measured platelet GPIb by using 6D1; platelet P selectin by using CD62P; platelet GPIIb/IIIa by using CD41; platelet factor X and platelet Annexin V by using reagents from R&D Systems (Minneapolis, MN); platelet fibrinogen with a reagent from Dako Cytomation (Carpinteria, CA); platelet microparticles identified by CD41 phycoerythrin and light scatter; and red blood cell Annexin V by the Coulter Epics XL (Beckman Coulter, Hialeah, FL).3

The thromboelastogram measurement was made in the blood products treated with 0.2 mol/L CaCl2 (Thrombelastogram Hemostasis Analyzer 500, Hemoscope Corp., Miles, IL).4–6 The R time measurement in the thromboelastogram was determined as the period of time that the blood was placed in the thromboelastogram analyzer until fibrin formation occurred.

Statistical analyses were performed with statistical software (SAS Institute, Cary, NC). Paired t tests were performed. A value of p < 0.05 was considered to indicate statistical significance. Means ± SD are reported.

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RESULTS

Table 1 shows that the R time was significantly (p < 0.05) reduced in the presence of poly-N-acetyl glucosamine fibers for PPP, PRP, and PRP plus red blood cells. Table 2 shows that the platelet Annexin V-binding level was not significantly increased in the presence of p-GlcNAc for PPP, PRP, and PRP plus red blood cells (WB1 with a hematocrit of 45%, WB2 with a hematocrit of 35%, and WB3 with a hematocrit of 20%).

Table 2 shows that platelet factor X binding was not significantly increased in the presence of p-GlcNAc for PPP, PRP, or PRP plus red blood cells. Table 2 also shows that platelet microparticles were not significantly increased in the presence of p-GlcNAc for PPP, PRP, or PRP plus red blood cells.

Table 3 shows that p-GlcNAc did not significantly increase red blood cell Annexin V binding in PRP plus red blood cells. Table 4 shows that in PRP plus red blood cells without p-GlcNAc and CaCl2, thromboxane B2 increased slightly as the hematocrit values increased; the higher the hematocrit, the higher the thromboxane B2 levels after incubation at 37°C for 30 minutes.

In the presence of poly-N-acetyl glucosamine fibers with or without CaCl2, the thromboxane B2 level increased in PRP. In the absence of CaCl2, both the hematocrit and p-GlcNAc were associated with increases in the thromboxane B2 level (Table 4). The increase in thromboxane B2 produced by p-GlcNAc in PRP was greater in the presence of CaCl2 than in the absence of CaCl2 (Table 3).

Figure 1 shows that p-GlcNAc in the presence of red blood cells and in the absence of CaCl2 reduced the fibrinogen level in PRP. When p-GlcNAc was added to PRP and red blood cells with a hematocrit of 45%, the fibrinogen level was reduced by 40%; when p-GlcNAc was added to PRP with a hematocrit of 35%, fibrinogen was reduced by 20%; and when p-GlcNAc was added to PRP with a hematocrit of 20%, fibrinogen was reduced by 15%. In the absence of CaCl2, no significant increases were observed in the prothrombin fragment 1.2 and D-dimer levels in PPP plus poly-N-acetyl glucosamine fibers, PRP plus p-GlcNAc, or PRP plus red blood cells plus p-GlcNAc after 30 minutes of incubation at 37°C.

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DISCUSSION

The addition of poly-N-acetyl glucosamine fiber slurry to PRP produced a significant reduction in the R time measurement in the thromboelastogram, slightly increased the production of thromboxane A2 by the platelets, slightly increased the level of platelet factor X, slightly increased the platelet level of Annexin V, and slightly increased the platelet microparticles. All of these measurements indicated activation of the platelets by the poly-N-acetyl glucosamine fiber slurry.

The addition of poly-N-acetyl glucosamine fiber material to PRP plus red blood cells produced a reduction in the R time measurement in the thromboelastogram, a slight increase in the production of thromboxane A2 by the platelets, a slight increase in platelet factor X, an increase in platelet Annexin V binding, and a slight increase in platelet microparticles. These findings suggest that the red blood cells augmented the activation of platelets by the poly-N-acetyl glucosamine slurry.

In the absence of CaCl2, the addition of poly-N-acetyl glucosamine fiber slurry to PRP plus red blood cells decreased the plasma fibrinogen level without increasing the prothrombin fragment 1.2 or D-dimer levels. These findings demonstrated that poly-N-acetyl glucosamine fiber material affects the clotting of blood by activation of the platelets.

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REFERENCES

1. Rylatt DB, Blake AS, Cottis LE, et al. An immunoassay for human D dimer using monoclonal antibodies. Thromb Res. 1983;31:767–778.
2. Feingold HM, Pivacek LE, Melaragno AJ, Valeri CR. Coagulation assays and platelet aggregation patterns in human, baboon, and canine blood. Am J Vet Res. 1986;47:2197–2199.
3. Barnard MR, MacGregor H, Ragno G, et al. Fresh, liquid-preserved, and cryopreserved platelets: adhesive surface receptors and membrane procoagulant activity. Transfusion. 1999;39:880–888.
4. Chandler WL. The thromboelastograph and the thromboelastograph technique. Semin Thromb Hemost. 1995;21:1–6.
5. Traverso CI, Caprini JA, Arcelus J, Arcelus IM. Thromboelastographic modifications induced by intravenous and subcutaneous heparin administration. Semin Thromb Hemost. 1995;21:53–58.
6. Summaria L. Thromboelastographic study of fibrinolytic agents. Semin Thromb Hemost. 1995;21:63–71.
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EDITORIAL COMMENT

The accompanying articles were presented during a symposium at the Brigham and Women’s Hospital, held on February 25, 2003. Considered together, the animal and clinical data indicate that poly-N-acetyl glucosamine, a U.S. Food and Drug Administration–approved agent manufactured by Marine Polymer Technologies, Inc., is effective in hemostasis whether applied by injection about gastric varices, topically against bleeding surfaces of spleen or liver, against major wounds of the abdominal aorta, or somewhat more remotely from the vascular injury, that is, on cardiac catheterization sites in the groin. These studies represent data from animal and human observations. Most of the studies were conducted in a blinded fashion with proper controls. The only difficulty with controlling the poly-N-acetyl glucosamine patch was that it often adhered with somewhat more tenacity than the control cellulose or deacetylated patches. Thus, some of the patch effects could be mechanical, although in most studies, this appeared to be a relatively insignificant action.

What makes these results even more striking is that positive hemostasis was achieved, even in the presence of full heparinization. The mechanism of action could still be by polymerization of fibrinogen by its contact with either activated platelets or red cells. That poly-N-acetyl glucosamine is a potent inducer of clot formation has been conclusively shown in several articles. However, to document the role of clotting in the clinical studies, it will be necessary to quantitate fibrin formation in the region of the vascular or solid organ injury.

That clotting factors may not be necessary to achieve hemostasis with poly-N-acetyl glucosamine was shown in an in vitro study of aortic injury. Data indicate that an endothelium-dependent vasoconstrictor is produced, which makes possible the induction of hemostasis by means of vascular contraction. The vasoconstrictor appears to be endothelin, a belief based largely on the observed ability of receptor antagonists to nullify the action of poly-N-acetyl glucosamine. However, neither enzyme-linked immunosorbent assay nor immunohistochemistry has been able to show a reduction in big endothelin or an increase in levels of endothelin at the site of vascular injury. This may be related to problems with antibody binding. These clinical and preclinical data support a potential role for poly-N-acetyl glucosamine in hemostasis in a variety of medical and surgical conditions and particularly in trauma.

Herbert B. Hechtman, MD

C. Robert Valeri, MD

Brigham and Women's Hospital

75 Francis Street

Boston, MA 02115

hhechtman@partners.org

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Keywords:

Platelet surface markers; Red blood cells; Phosphatidylserine; Thromboelastogram

© 2004 Lippincott Williams & Wilkins, Inc.