The mitochondrial permeability transition pore provides a key to the diagnosis and treatment of traumatic brain injury.

The pathological consequences of traumatic head injury result largely from the opening of the mitochondrial permeability transition pore (mPTP). The mPTP opens due to a decrease in brain phosphorylation energy resulting in a further decrease in brain ATP production and a measurable increase in brain heat generation and temperature. The increase in brain temperature can be measured transcranially by near infrared spectroscopy which can be used to diagnose traumatic brain injury (TBI) and to monitor treatment. Effective therapy of TBI can be achieved by closure of the mPTP by administration of cyclosporine A or by oral administration of ketone body esters. While ketosis has previously been known to prevent damage from TBI, the availability of oral ketone esters presents the first practical modality of achieving therapeutic levels of ketone bodies.

mRDH bandage for surgery and trauma: data summary and comparative review.

BACKGROUND: Bleeding often poses significant life-threatening situations to surgeons. After trauma, a one-third of civilian casualties and one-half of combat casualties die as a result of exsanguination. Recent advances have provided promising new hemostatic dressings that are applied directly to severely bleeding wounds in the pre-hospital period. METHODS: The modified Rapid Deployment Hemostat (mRDH) trauma/surgery bandage, containing fully acetylated, diatom-derived, poly-N-acetyl-glucosamine fibers, has a unique multifactorial hemostatic action that incorporates vasoconstriction, erythrocyte agglutination, and platelet and RBC activation. RESULTS: Animal studies have shown that the mRDH bandage quickly and completely stops both venous and arterial bleeding, even in the presence of a coagulopathy. A prospective study in humans is in accord with these findings. CONCLUSION: The mRDH trauma/surgery bandage was able to increase survival of patients after high-grade liver trauma with an associated coagulopathy. Additional clinical studies support this result.

Poly-N-acetyl glucosamine fibers accelerate hemostasis in patients treated with antiplatelet drugs.

BACKGROUND: Nanofibers consisting of poly-N-acetyl glucosamine (pGlcNAc), as the functional component of products for surface hemostasis, have been shown to activate platelets and thereby the clotting mechanism. The nanofiber-activated platelets provide a catalytic surface for acceleration of the intrinsic coagulation cascade, thrombin generation, and fibrin polymerization. METHODS: Thromboelastographic analysis was undertaken to study the role of the pGlcNAc nanofibers in platelet activation and acceleration of fibrin polymerization. Thromboelastographic studies were performed without added activators of coagulation. RESULTS: The pGlcNAc nanofibers were found to accelerate fibrin polymerization in whole blood and platelet-rich plasma. Treatment with eptifibatide (an inhibitor of the platelet GPIIbIIIa receptor) and corn trypsin inhibitor inhibited clotting of whole blood and platelet-rich plasma. The inhibition was reversed by treatment with pGlcNAc nanofibers. Inhibition was not observed after treatment with aspirin alone, MRS2359 (platelet ADP receptor inhibitor), or by a combination of aspirin and MRS2359. The pGlcNAc nanofibers accelerate clotting in normal blood treated with aspirin and MRS2359. Clopidogrel (Plavix) and aspirin did not affect the kinetics of pGlcNAc-mediated fibrin polymerization in blood from patients treated with antiplatelet drugs compared with nontreated blood. CONCLUSIONS: These results provide evidence that pGlcNAc nanofibers activate platelets and accelerate the clotting of blood, and on how best to achieve surface hemostasis when patients are coagulopathic because of shock and/or to treatment with antiplatelet drugs.

Poly-N-acetyl glucosamine fibers induce angiogenesis in ADP inhibitor-treated diabetic mice.

BACKGROUND: It has been previously demonstrated that short-fiber poly-N-acetyl-glucosamine (sNAG) nanofibers specifically interact with platelets, are hemostatic, and stimulate diabetic wound healing by activating angiogenesis, cell proliferation, and reepithelialization. Platelets play a significant physiologic role in wound healing. The influence of altered platelet function by treatment with the ADP inhibitor Clopidogrel (CL) on wound healing and the ability of sNAG to repair wounds in diabetic mice treated with CL were studied. METHODS: Dorsal 1 cm2 skin wounds were excised on genetically diabetic 8-week to 12-week-old, Lep/r-db/db male mice, and wound healing kinetics were determined. Microscopic analysis was performed for angiogenesis (PECAM-1) and cell proliferation (Ki67). Mice were either treated with CL (P2Y12 ADP receptor antagonist, CL) or saline solution (NT). CL wounds were also treated with either a single application of topical sNAG (CL-sNAG) or were left untreated (CL-NT). RESULTS: CL treatment did not alter wound healing kinetics, while sNAG induced faster wound closure in CL-treated mice compared with controls. CL treatment of diabetic mice caused an augmentation of cell proliferation and reduced angiogenesis compared with nontreated wounds. However, sNAG reversed the effects of CL on angiogenesis and partially reversed the effect on cell proliferation in the wound beds. The sNAG-treated wounds in CL-treated mice showed higher levels of cell proliferation and not did inhibit angiogenesis. CONCLUSIONS: CL treatment of diabetic mice decreased angiogenesis and increased cell proliferation in wounds but did not influence macroscopic wound healing kinetics. sNAG treatment did not inhibit angiogenesis in CL-treated mice and induced faster wound closure; sNAG technology is a promising strategy to facilitate the healing of complex bleeding wounds in CL-treated diabetic patients.

Poly-N-acetyl glucosamine fibers are synergistic with vacuum-assisted closure in augmenting the healing response of diabetic mice.

BACKGROUND: Vacuum-assisted closure (VAC) has become the preferred modality to treat many complex wounds but could be further improved by methods that minimize bleeding and facilitate wound epithelialization. Short fiber poly-N-acetyl glucosamine nanofibers (sNAG) are effective hemostatic agents that activate platelets and facilitate wound epithelialization. We hypothesized that sNAG used in combination with the VAC device could be synergistic in promoting wound healing while minimizing the risk of bleeding. METHODS: Membranes consisting entirely of sNAG nanofibers were applied immediately to dorsal excisional wounds of db/db mice followed by application of the VAC device. Wound healing kinetics, angiogenesis, and wound-related growth factor expression were measured. RESULTS: The application of sNAG membranes to wounds 24 hours before application of the VAC device was associated with a significant activation of wounds (expression of PDGF, TGFß, EGF), superior granulation tissue formation rich in Collagen I as well as superior wound epithelialization (8.6% ± 0.3% vs. 1.8% ± 1.1% of initial wound size) and wound contraction. CONCLUSIONS: The application of sNAG fiber-containing membranes before the application of the polyurethane foam interface of VAC devices leads to superior healing in db/db mice and represents a promising wound healing adjunct that can also reduce the risk of bleeding complications.

An approach to prevent the severe adverse events associated with transfusion of FDA-approved blood products.

There have been several retrospective studies reporting severe adverse events of mortality and morbidity associated with blood transfusions. Mortality and morbidity associated with posttransfusion infection, transfusion related acute lung injury (TRALI), and systemic inflammatory response syndrome (SIRS) have been reported in patients undergoing cardiac surgery, after massive transfusions for severe traumatic injuries, and after transfusions for elective and emergency indications. After 35 days of storage at 4 degrees C in additive solutions, RBC have 24-h posttransfusion survival values of 75% but do not function satisfactorily. For RBC to function satisfactorily shortly after transfusion, they should be stored at 4 degrees C for no more than 2 weeks. Yet while the FDA requires a 24-h posttransfusion survival value of 75%, there is no requirement for the function of the transfused RBC. It has been shown that red blood cells that circulate and function immediately or shortly after transfusion exert a very important hemostatic effect to reduce the bleeding time and nonsurgical blood loss in anemic and thrombocytopenic patients. Greater restoration of hemostasis is seen with viable and functional RBC transfusions than with platelets or plasma even though the platelets and plasma proteins may have satisfactory viability and function. The length of storage of the blood products affects their survival and function and the transfusion of nonviable compatible RBC, antibodies to granulocytes and WBC HLA antigens and biologically active substances affects the patient's clinical outcome. One of the easiest ways to prevent the severe adverse events that have been observed is to ensure that the transfused blood products survive and function at an optimum level and that the levels of antibodies to granulocytes and WBC HLA antigens and biologically active substances are eliminated or reduced. The best way to ensure this is to store liquid-preserved leukoreduced human red blood cells at 4 degrees C in additive solutions for no more than 2 weeks and leukoreduced platelets at room temperature for no more than 2 days. These liquid-preserved blood products can be used in conjunction with frozen RBC, platelets, and plasma stored in -80 degrees C mechanical freezers and will avoid the need for fresh whole blood and prevent the severe adverse events associated with the transfusion of blood products.

Quiescent platelets stimulate angiogenesis and diabetic wound repair.

INTRODUCTION: Platelets partake in hemostasis, wound healing, and tumor growth. Although platelet-rich-plasma (PRP) has been used in surgery for several years, its mechanism of action and application methods are still poorly characterized. MATERIALS AND METHODS: A single unit of human platelets obtained by plateletpheresis was diluted in plasma and divided into three equal volumes. One volume was stored at room temperature as fresh platelets (RT), another volume was frozen by storage at -80 degrees C (FZ), and the third volume was frozen at -80 degrees C with 6% DMSO (FZ6). Plasma (PL) was used as control. Using flow cytometry, platelets were tested for platelet glycoprotein GPIb and annexin V binding, as survival and activation markers, respectively. Hemostatic function was assessed by thromboelastometry. In vivo, platelets were topically applied on 1 cm,(2) full-thickness wounds on db/db mice (n = 10/group) and healing was staged microscopically and macroscopically. RESULTS: All platelet preparations showed hemostatic ability. RT platelets were GPIb positive (nonactivated-quiescent platelets) and stimulated angiogenesis by threefold, and cell proliferation by fourfold in vivo. FZ platelets were positive for annexin V, indicating activated platelets and, in vivo, increased only wound granulation. FZ6 platelets contained 30% nonactivated-quiescent and 50% activated platelets and stimulated granulation, angiogenesis, cell proliferation, and promoted re-epithelialization in vivo. CONCLUSIONS: Platelets showed distinct mechanisms to induce hemostasis and wound healing. Quiescent platelets are required to induce angiogenesis in vivo. Platelets stored at room temperature and frozen with 6% DMSO and stored at -80 degrees C achieved optimal wound healing in diabetic mice.

Non-classical processes in surface hemostasis: mechanisms for the poly-N-acetyl glucosamine-induced alteration of red blood cell morphology and surface prothrombogenicity.

It is well established that platelets and the intrinsic plasma coagulation pathway can be activated when blood contacts artificial surfaces. Experiments were performed to assess the effect of hemostatic poly-N-acetyl glucosamine (pGlcNAc) nanofibers on red blood cells. The pGlcNAc nanofibers, isolated from a marine diatom, interact with red blood cells (RBCs) to produce stomatocytes. The stomatocytes could be converted to echinocytes by treatment with echinocytic reagents, as measured by electron microscopy. Electrophoretic and Western blot analysis of RBC surface proteins demonstrated that pGlcNAc fibers were bound to band 3 of the RBC. An important and unique result of the interaction of RBCs with pGlcNAc fibers was the activation of the intrinsic coagulation cascade. This prothrombotic effect was associated with the presentation of phosphatidylserine on the outer layer of the surface membrane of nanofiber bound RBCs. The results demonstrate that RBCs can play a direct and important role in achieving surface hemostasis by accelerating the generation of thrombin, and add to the growing body of evidence that RBCs can strongly interact with hemostatic systems.

Effects of poly-N-acetyl glucosamine (pGlcNAc) patch on wound healing in db/db mouse.

BACKGROUND: Poly-N-acetyl glucosamine (pGlcNAc) nanofiber-based materials, produced by a marine microalga, have been characterized as effective hemostatic agents. In this study, we hypothesized that a pGlcNAc fiber patch may enhance wound healing in the db/db mouse. METHODS: pGlcNAc patches were applied on 1-cm, full-thickness, skin wounds in the db/db mouse model. Wounds (n = 15 per group) were dressed with a pGlcNAc nanofiber patch for 1 hour, 24 hours, or left untreated. After the application time, patches were removed and wounds were allowed to heal spontaneously. The rate of wound closure was evaluated by digital analysis of unclosed wound area as a function of time. At day 10, wounds (n = 7 per group) were harvested and quantified with immunohistochemical markers of proliferation (Ki-67) and vascularization (platelet endothelial cell adhesion molecule). RESULTS: Wounds dressed with pGlcNAc patches for 1 hour closed faster than control wounds, reaching 90% closure in 16.6 days, 9 days faster than untreated wounds. Granulation tissue showed higher levels of proliferation and vascularization after 1-hour treatment than the 24-hour and left-untreated groups. Foreign body reaction to the material was not noted in applications up to 24 hours. DISCUSSION: In addition to its hemostatic properties, the pGlcNAc material also appears to accelerate wound closure in healing-impaired genetically diabetic mice. This material, with its combination of hemostatic and wound healing properties, has the potential to be effective agent for the treatment of complicated wounds.

The effects of preserved red blood cells on the severe adverse events observed in patients infused with hemoglobin based oxygen carriers.

The severe adverse events observed in patients who received hemoglobin based oxygen carriers (HBOCs) were associated with the Ringer's D.L lactate resuscitative solution administered and to the excipient used in the HBOCs containing Ringer's D,L lactate and the length of storage of the preserved RBC administered to the patient at the time that the HBOCs were infused. This paper reports the quality of the red blood cells preserved in the liquid state at 4 degrees C and that of previously frozen RBCs stored at 4 degrees C with regard to their survival, function and safety. Severe adverse events have been observed related to the length of storage of the liquid preserved RBC stored at 4 degrees C prior to transfusion. The current methods to preserve RBC in the liquid state in additive solutions at 4 degrees C maintain their survival and function for only 2 weeks. The freezing of red blood cells with 40% W/V glycerol and storage at -80 degrees C allows for storage at -80 degrees C for 10 years and following thawing, deglycerolization and storage at 4 degrees C in the additive solution (AS-3, Nutricel) for 2 weeks with acceptable 24 hour posttransfusion survival, less than 1% hemolysis, and moderately impaired oxygen transport function with no associated adverse events. Frozen deglycerolized RBCs are leukoreduced and contain less than 5% of residual plasma and non-plasma substances. Frozen deglycerolized RBCs are the ideal RBC product to transfuse patients receiving HBOCs.

Basic science focus on blood substitutes: a summary of the NHLBI Division of Blood Diseases and Resources Working Group Workshop, March 1, 2006.

In March 2006, a workshop sponsored by the National Heart, Lung, and Blood Institute was convened to identify the role of basic science research in clarifying issues that are impeding progress in the development of hemoglobin-based oxygen carrying (HBOC) solutions. These discussions resulted in a consensus that, although HBOCs have shown clinical promise, various side effects have inhibited further development and regulatory approval, with cardiovascular events being of particular concern. As a consequence, workshop participants focused on formulating research recommendations to better understand and mitigate these side effects. In addition, several important corollary issues were identified, including better understanding of the impact of HBOC infusion on human physiology; the need for rapid, noninvasive methods for the measurement of tissue oxygenation in human patients to better inform transfusion decisions; further investigation of routes and consequences of hemoglobin metabolism; optimization of clinical protocols for HBOC use; and assessment of the impact of HBOC formulation excipients. Also discussed was the possibility and desirability of developing new HBOCs with improved characteristics, such as prolonged functional intravascular persistence, greater stability, and a decreased propensity to generate reactive oxygen species. One practical limitation in this area is the consistent availability of pure, well-characterized HBOC solutions for the research community. This communication summarizes the opinion of workshop participants on these issues and concludes with a list of specific recommended areas of research that could positively impact the development of blood substitutes.

Nonsurgical bleeding diathesis in anemic thrombocytopenic patients: role of temperature, red blood cells, platelets, and plasma-clotting proteins.

Research at the Naval Blood Research Laboratory (Boston, MA) for the past four decades has focused on the preservation of red blood cells (RBCs), platelets (PLTs), and plasma-clotting proteins to treat wounded servicemen suffering blood loss. We have studied the survival and function of fresh and preserved RBCs and PLTs and the function of fresh and frozen plasma-clotting proteins. This report summarizes our peer-reviewed publications on the effects of temperature, RBCs, PLTs, and plasma-clotting proteins on the bleeding time (BT) and nonsurgical blood loss. The term nonsurgical blood loss refers to generalized, systemic bleeding that is not corrected by surgical interventions. We observed that the BT correlated with the volume of shed blood collected at the BT site and to the nonsurgical blood loss in anemic thrombocytopenic patients after cardiopulmonary bypass surgery. Many factors influence the BT, including temperature; hematocrit (Hct); PLT count; PLT size; PLT function; and the plasma-clotting proteins factor (F)VIII, von Willebrand factor, and fibrinogen level. Our laboratory has studied temperature, Hct, PLT count, PLT size, and PLT function in studies performed in non-aspirin-treated and aspirin-treated volunteers, in aspirin-treated baboons, and in anemic thrombocytopenic patients. This monograph discusses the role of RBCs and PLTs in the restoration of hemostasis, in the hope that a better understanding of the hemostatic mechanism might improve the treatment of anemic thrombocytopenic patients. Data from our studies have demonstrated that it is important to transfuse anemic thrombocytopenic patients with RBCs that have satisfactory viability and function to achieve a Hct level of 35 vol percent before transfusing viable and functional PLTs. The Biomedical Excellence for Safer Transfusion (BEST) Collaborative recommends that preserved PLTs have an in vivo recovery of 66 percent of that of fresh PLTs and a life span that is at least 50 percent that of fresh PLTs. Their recommendation does not include any indication that preserved PLTs must be able to function to reduce the BT and reduce or prevent nonsurgical blood loss. One of the hemostatic effects of RBC is to scavenge endothelial cell nitric oxide, a vasodilating agent that inhibits PLT function. In addition, endothelin may be released from endothelial cells, a potent vasoconstrictor substance,to reduce blood flow at the BT site. RBCs, like PLTs at the BT site, may provide arachidonic acid and adenosine diphosphate to stimulate the PLTs to make thromboxane, another potent vasoconstrictor substance and a PLT-aggregating substance. At the BT site, the PLTs and RBCs are activated and phosphatidyl serine is exposed on both the PLTs and the RBCs. FVa and FXa, which generate prothrombinase activity to produce thrombin, accumulate on the PLTs and RBCs. A Hct level of 35 vol percent at the BT site minimizes shear stress and reduces nitric oxide produced by endothelial cells. The transfusion trigger for prophylactic PLT transfusion should consider both the Hct and the PLT count. The transfusion of RBCs that are both viable and functional to anemic thrombocytopenic patients may reduce the need for prophylactic leukoreduced PLTs, the alloimmunization of the patients, and the associated adverse events related to transfusion-related acute lung injury. The cost for RBC transfusions will be significantly less than the cost for the prophylactic PLT transfusions.