Harvard Symposium on the Clinical Efficacy and Hemostatic Mechanism of Action of Poly-N-Acetyl Glucosamine

Figure 2.  Comparative efficacy of SyvekPatch and other products.
Figure 3.  Vasoconstriction by SyvekPatch material at vascular access site (Ikeda, Y. et al)
Figure 1.  3D-Structure of SyvekPatch Fiber Component
Figure 4.  Red blood cells aggregate on surface of SyvekPatch.
Figure 5.  Platelet contact with SyvekPatch results in platelet cell activation.
Author(s): 

Joshua A. Hirsch, MD, Massachusetts General Hospital, Assistant Professor Harvard Medical School, Boston, Massachusetts

This symposium provided a forum for several prominent clinicians and scientists. They presented summaries of their laboratory and clinical data on the use and hemostatic mechanism of poly-N-acetyl glucosamine, the material that makes up the SyvekPatch in its entirety.

Dr. S.F. Khuri of Harvard Medical School presented the results of a blinded, randomized, placebo-controlled clinical trial to evaluate the efficacy of the SyvekPatch in improving hemostasis in patients undergoing cardiac catheterization. Also, data were presented on the details of the interaction of blood cells with poly-N-acetyl glucosamine that led to an understanding of the hemostatic mechanism. In this article, I’ll discuss some of the results presented at the meeting, which include:

Aspects of the chemical and structural properties of the polymer;

Comparisons of SyvekPatch with chitosan-based products and other hemostats in pre-clinical studies;

Advances in the formulation and utilization of the RDH Bandage (a hemostatic bandage formulated from pGlcNAc) in a number of pre-clinical trauma applications;

Comprehensive data on the hemostatic mechanism of action of poly-N-acetyl glucosamine.

Structure of Poly-N-acetyl Glucosamine in SyvekPatch

Poly-N-acetyl glucosamine (pGlcNAc) is a polysaccharide derived from a marine microalga in a unique semi-crystalline structural form. Materials formulated from pGlcNAc have been found to be effective in achieving hemostasis in cardiac catheterization, surgical procedures, and in trauma. Dr. John Vournakis of Marine Polymer Technologies described pGlcNAc molecular structure as that of a fully-acetylated polymer consisting of approximately 2000 N-acetyl glucosamine residues with a molecular weight of approximately 3 X 106 Daltons. It is fully biocompatible and can be formulated into films, sponges, hydrogels, microspheres, and fibers. Its unique structure is key to its hemostatic properties (see Figure 1). Other polymers containing N-acetyl glucosamine, such as chitin, chitosan, and hyaluronic acid, do not have the same structure, and as a result, do not have significant hemostatic activity.

Preclinical Studies: Coagulopathic Animal Models

Dr. Steven D. Schwaitzberg and colleagues of the New England Medical Center in Boston presented studies on the hemostatic efficacy of SyvekPatch and four currently available products:

Actifoam (C.R. Bard, Inc., Murray Hill, NJ)

Surgicel (J&J Medical, Arlington, Texas)

Two chitosan products

- Clo-Sur P.A.D. (Scion Cardio-Vascular, Miami, FL)

- Chito-Seal (Abbott Laboratories, Abbott Park, IL)

The studies were in three coagulopathic animal models (bleeding was induced by splenic laceration):

In systemically heparinized pigs, SyvekPatch was significantly more effective than Actifoam, Surgicel, Clo-Sur P.A.D., or Chito-Seal (see Figure 2).

In hemophiliac dogs, SyvekPatch was significantly more effective than Surgicel.

In hypothermic rabbits, the SyvekPatch was as effective at a low temperature (29°C) as at 37°C.

The complete set of data obtained from the models indicates that SyvekPatch is effective at controlling bleeding in animals that have either experimentally induced or genetic coagulopathic conditions.

Preclinical Studies: Animal Trauma Models of Hemorrhage

Dr. R. J. Connolly and colleagues of the New England Medical Center compared the RDH Bandage (a trauma hemostatic bandage formulated from pGlcNAc) with:

Gauze pads;

The standard issue U.S. Army First Aid Field Bandage;

TachoComb® (Nycomed Austria, Linz, Austria).

The purpose of the comparison was to measure each product’s control of bleeding in three swine trauma models of hemorrhage:

A lower extremity injury model involving skin, muscle, bone and femoral artery injury simulating battlefield wounds;

Two aortic models that use vertical incisions or circular punches to produce lethal injuries.

In all three models, the RDH Bandage was found to be significantly superior to the other three products in controlling hemorrhage.

Dr. S. M. Cohn and colleagues of the University of Miami School of Medicine investigated whether the RDH Bandage could achieve hemostasis when used as an adjunct to standard abdominal packing after severe liver injury in coagulopathic swine. The pigs received an isovolemic 45% blood volume replacement with cold 6% hetastarch. The core body temperature was maintained at 33°-34°C, and a hypocoagulopathic state was documented by thromboelastography. Liver injury was induced by avulsion of the left lateral hepatic lobe. Results showed that use of the RDH Bandage, in addition to standard abdominal packing, reduced blood loss and fluid requirements, leading to improved survival compared to controls.

Clinical Studies: Bowel Surgery

Dr. D. J. Cole and colleagues of the New England Medical Center compared SyvekPatch with Surgicel. The comparison was the first human trial with Surgicel for the control of bleeding induced in segments of small bowel that were about to be removed from patients undergoing elective bowel surgery. SyvekPatch was more effective than Surgicel in this human study.

Clinical Studies: Cardiac Catheterization

Hemostasis following arterial puncture for cardiac catheterization is currently achieved by applying pressure to the puncture site. In a blinded, randomized, placebo-controlled clinical trial, Dr. S. F. Khuri and colleagues of the Harvard Medical School compared the efficacy of a placebo-patch and SyvekPatch to control bleeding at the femoral insertion site of patients who had undergone cardiac catheterization. This is the first blinded, randomized, placebo-controlled clinical trial to evaluate the efficacy of poly-N-acetyl glucosamine in improving hemostasis in patients undergoing cardiac catheterization.

At the conclusion of their catheterization procedure, patients were randomly assigned to have either a 3x3 cm placebo-patch (n=17) or SyvekPatch (n=16) topically placed at the femoral insertion site, with a mechanical pressure clamp applied over it. The amount of pressure applied was measured using a fluid-filled balloon connected to a pressure transducer. The clamp was loosened at 5-minute intervals to check for hemostasis at the site, but the patch was not disturbed. A pre-catheterization bleeding time was also performed. No differences were seen in the pre-catheterization hematocrit, bleeding time, and activated clotting time. Additionally, no differences between the two groups were noted in the following variables:

Age

BSA

Pre-cath anticoagulation therapy

Arm temperature

Prothrombin time

Partial thromboplastin time

Red blood cells count

White blood cell count

Platelet count

There was also no difference in the clamp pressure applied to the femoral arterial puncture site at the end of the catheterization procedure.

The application of SyvekPatch in this blinded, randomized, placebo-controlled clinical trial showed a statistically significant decrease in closure time due to rapid hemostasis at the arterial puncture site in patients undergoing cardiac catheterization. It is remarkable that in spite of the relatively small number of human subjects in the study, statistical significance was achieved by the SyvekPatch.

pGlcNAc Gels for Control of Variceal Bleeding

In a double-blind study in his classic dog model of variceal bleeding, Dr. D. M. Jensen and colleagues of the UCLA School of Medicine/VA GLAHS and the VA Greater Los Angeles Healthcare System in Los Angeles compared pGlcNAc-derived gel materials in the treatment of bleeding gastric varices with:

Glue (n-butyl cyanoacrylate);

A sclerosant mixture (3.3% ethanolamine and 32% alcohol);

Control saline.

Each dog had pre-hepatic portal hypertension and five or six moderate- to large-sized gastric varices. The dogs were heparinized, and bleeding was induced with a 19-gauge needle. The results were evaluated by endoscopy after seven days.

The use of glue was associated with a high rate of primary hemostasis, but it was also associated with the highest ulceration rate and no change in the size of gastric varices.

The sclerosant mixture was associated with a low rate of primary hemostasis, a moderately high ulceration rate, and a moderate reduction in the size of gastric varices.

The pGlcNAc gels, at concentrations of

1% and 3%, were associated with:

- high rates of primary and five-minute hemostasis;

- significant reduction in the size of gastric varices;

- a low ulceration rate.

Application of pGlcNAc gels appears very promising for hemostasis of bleeding gastric varices and reduction of their size.

Studies of the Poly-N-acetyl Glucosamine (pGlcNAc)
Hemostatic Mechanism

a. Vasoconstriction

Dr. A. M. Lefer of the Jefferson Medical College in Philadelphia investigated the mechanism of vasoconstriction by pGlcNAc in isolated rat aortic rings.

pGlcNAc produced a concentration-dependent contraction of the rings over a range of 14 to 140 µg/ml (see Figure 3; Ikeda Y et al). Neither a deacetylated derivative of pGlcNAc nor the chemically related macromolecules chitin and chitosan produced vasoconstriction, indicating that the action is specific to pGlcNAc. In fact, both chitin and chitosan caused vaso-dilation in the aortic rings. Vasoconstriction induced by pGlcNAc was totally abolished in de-endothelialized aortic rings. Pretreatment with the endothelin ETA receptor antagonist JKC-301 significantly diminished pGlcNAc-induced vasoconstriction.

These results provide evidence that pGlcNAc causes significant vasoconstriction in isolated rat aortic rings by an endothelium-dependent mechanism, acting in part by enhancement of the release of endothelin-1 from endothelial cells.

Dr. H. B. Hechtman and colleagues of Brigham and Women’s Hospital in Boston tested the hypothesis that one mechanism of pGlcNAc action during hemostasis is to induce release of a local vasoconstrictor, such as endothelin-1, leading to contraction of the smooth muscle of the blood vessel wall and closure of the laceration.

A segment of rat aorta was removed and tied with ligatures at both ends as it was flushed with saline. A cannula was inserted into the segment and connected to a reservoir of saline. The aorta was punctured with a needle, a control or pGlcNAc patch was placed on the injury, and the time taken for the reservoir to empty was recorded. The emptying time was longer with the pGlcNAc patch than with the control patch, indicating that the pGlcNAc patch produced constriction of the puncture wound. Treatment with endothelin-1 receptor antagonists reduced the emptying time to control values.

It was concluded that pGlcNAc is an effective topical hemostatic agent that acts by the release of endothelin-1. This action occurs in the absence of any formed elements of blood.

b. Red Blood Cell Aggregation

Dr. H. S. Thatte of the Harvard Medical School and the West Roxnbury V.A. Hospital tested the hypothesis that pGlcNAc induces hemostasis primarily by its interaction with blood cells at the wound site.

P-GlcNAc and various chemically modified pGlcNAc interactions with human and porcine blood cells (whole blood, red blood cells, and platelets) were evaluated by multiphoton imaging and spectrophotometry. The ability of pGlcNAc to induce rapid hemostasis in excised, injured human saphenous vein was demonstrated by multiphoton microscopy. The cell-surface receptors involved in interactions with pGlcNAc and the activation of blood cells in vitro were identified by fluorescence and immunological techniques.

It was concluded that pGlcNAc-induced thrombogenesis is mediated via ionic interactions and cell-surface receptors on various blood cell types. The hemostatic mechanism of p-GlcNAc is primarily due to its mechanistic interaction with, and then activation of, various blood cells that are encountered at the wound site (see Figure 4).

c. Platelet Aggregation, Activation and Clot Formation

Dr. T. H. Fischer and Dr. E. P. Merricks of the University of North Carolina at Chapel Hill studied the specific chemical and physical absorption processes involved in the interaction of platelets with pGlcNAc, leading to hemostasis.

Surface proteins of platelets were labeled with a membrane-impermeable biotin-NHS derivative and mixed with pGlcNAc polymers in the presence or absence of plasma. The interaction of platelets with pGlcNAc resulted in changes in shape and the extension of pseudopodia, as well as the presentation of surface fibrinogen and p-selectin from alpha granule pools (see Figure 5). Platelets bound to the pGlcNAc polymer to form pGlcNAc-fibrin-platelet macroaggregates. A wide variety of platelet surface proteins physically contacted and affinity-absorbed to the polymer.

pGlcNAc accelerated fibrin polymerization and participated in platelet-driven clot retraction of pGlcNAc-fibrin-platelet macroaggregates. The interaction of platelets with pGlcNAc was partially inhibited by GP IIb/IIIa antagonists, corn trypsin inhibitor, and heparin.

These results are consistent with a three-step mechanism of interaction between platelets and pGlcNAc fibers.

1. First, platelets physically bind directly to pGlcNAc and/or pGlcNAc-immobilized serum proteins (in an altered conformation), such as fibrinogen. The pGlcNAc-platelet interaction initiates a platelet activation response.

2. Second, pGlcNAc initiates the intrinsic (contact) coagulation factor pathway by activating factor XII, leading to thrombin generation and fibrin polymerization.

3. Finally, the resulting pGlcNAc-fibrin-platelet structures undergo platelet-mediated clot retraction.

d. Red Blood Cell Activation and Clot Formation

Dr. R. C. Valeri, Director of the Naval Blood Research Laboratory at the Boston University School of Medicine, studied the effect of pGlcNAc on blood clotting.

In the thromboelastogram, pGlcNAc reduced the time to initiate clot formation in platelet-poor plasma, platelet-rich plasma, and platelet-rich plasma supplemented with red blood cells. pGlcNAc increased the binding of annexin V to platelets, increased platelet microparticles, and surprisingly, increased the binding of annexin V to red blood cells. In platelet-rich plasma supplemented with red blood cells, pGlcNAc increased p-selectin.

It was concluded that pGlcNAc promotes blood clotting by activating plasma clotting proteins, platelets, and red blood cells.

Conclusions

The Harvard Medical School Symposium on Clinical Activity and Mode of Action of the Hemostatic Agent Poly-N-Acetyl Glucosamine (pGlcNAc) demonstrated the versatility of possible surgical applications of materials consisting of the natural polymer. It seems to have excellent properties that make it an attractive basis for the development of a variety of hemostatic products.

The mechanism of action of the polymer is closely linked to its physical structure. It allows for specific receptor-based interactions with both platelets and red blood cells. These interactions activate several aspects of the physiological and biochemical cascades, ultimately resulting in hemostasis. It is likely that a number of useful products will be developed during the coming years that take advantage of the uniqueness of this natural biopolymer.

References: 
<B>Recommended Reading</B>1. Nader RG, Garcia MD, Drushal K, Pesek T. Clinical Evaluation of the SyvekPatch® in Consecutive Patients Undergoing Interventional, EPS and Diagnostic Cardiac Catheterization Procedures. J Invas Cardiol 2002;14:305–307.2. Cole DJ, Connolly RJ, Chan MW, Swaitzberg SD, et al. A Pilot Study Evaluating the Efficacy of a Fully Acetylated Poly-_-1_4-N-Acetyl glucosamine Membrane Formulation as a Topical Hemostatic Agent. Surgery 1999;126:510–517.3. Kulling D, Vournakis JN, Woo S, et al. Endoscopic Injection of Bleeding Esophageal Varices with a Poly-_-1_4-N-Acetylglucosamine Gel Formulation in the Canine Portal Hypertension Model. Gastrointestinal Endoscopy 1999;49:764–771.4. Chan MW, Schwaitzberg SD, Demcheva M, et al. Pre-Clinical Comparison of p-GlcNAc with Absorbable Collagen (Actifoam) and Fibrin Sealant for Achieving Hemostasis in a Swine Model of Splenic Hemorrhage. J Trauma 2000;48:454–458.5. Ikeda Y, Young LH, Demcheva M, et al.Vascular Effects of Poly-N-Acetylgucosamine in Isolated Rat Aortic Rings. Journal of Surgical Research 2002;102: 215–220.6. Thomas E. Early ambulation following cardiac catheterization using the SyvekPatch®. Cath Lab Digest Nov 2002.7. Ebersole D. A novel approach to vascular access closure following cardiac catheterization procedure. Cath Lab Digest Mar 2001.8. Glowacki J, Dooley B. The use of SyvekPatch after diagnostic cardiac catheterization to reduce the cost of post procedure recovery and increase patient satisfaction. Cath Lab Digest Sept 2001.9. Hirsch J. Proceedings American Society of Neuroradiology 39th Annual Meeting. Boston, Massachusetts 2001.

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