OBJECTIVE To examine the safety and feasibility of using lyophilized platelets (LYO) and fresh platelet concentrate (FRESH) in bleeding thrombocytopenic dogs. DESIGN Preliminary prospective randomized clinical trial. SETTING Two private referral centers and 3 university teaching hospitals. ANIMALS Thirty-seven dogs with a complaint of hemorrhage associated with thrombocytopenia (platelet count <70 × 10(9) /L [70,000/μL], a hematocrit >15%, and that had received neither vincristine nor platelet-containing transfusions within 72 h of enrollment were studied. INTERVENTIONS Animals were randomized to receive LYO or FRESH, dosed according to weight. Physical examination, complete blood counts, and coagulation testing (prothrombin time and activated partial thromboplastin time) were performed at enrollment. Physical examinations were also performed immediately post transfusion, and at 1 and 24 h after transfusion. Complete blood counts were repeated immediately post transfusion and at 24 h. Collected data included bleeding score (BLS), response to transfusion, adverse reactions, hospitalization time, need for additional transfusions, survival to discharge, and 28-d survival. MEASUREMENTS AND MAIN RESULTS Twenty-two dogs received LYO and 15 received FRESH. There was no difference between groups in age, weight, BLS, platelet count, white blood cell count, hematocrit, or presence of melena. There was no difference between groups in transfusion reaction rates, the need for additional transfusions, 24-h BLS, hospitalization time, survival to discharge, or 28-d survival. CONCLUSIONS Transfusion of LYO was feasible and associated with a low transfusion reaction rate in this limited study of thrombocytopenic canine patients presenting with mild-to-severe hemorrhage. LYO were easy to use and provided storage advantages over FRESH. Further study of this product, including examination of efficacy and platelet life span, is warranted.

The experiments presented here were undertaken to determine if factor VIIa (rFVIIa, the Novo Nordisk product NovoSeven) will directly bind to rehydrated, lyophilized (RL) platelets for the formation of a catalytic surface with an enhanced ability to generate thrombin. The interaction between rFVIIa and the RL platelet surface was examined by measuring equilibrium and non-equilibrium binding of the coagulation factor to the cells and by following the effects of the surface modification on the kinetics of thrombin generation. The association of rFVIIa with RL platelets was rapid with saturation occurring within minutes. Disassociation was slow, with over half of the coagulation factor remaining bound after two hours. Densities of over one million molecules of rFVIIa per RL platelet were obtained when high concentrations of rFVIIa were incubated with RL platelets. Thrombin generation measurements showed that RL platelet-bound rFVIIa was catalytically active. Thus we can expect that RL platelets, which have been shown to effectively bind to sites of vascular injury, will localize rFVIIa to wounds for an increase in therapeutic index. These studies indicate that rFVIIa-RL platelets are worthy of preclinical and clinical development as an infusion agent for severe bleeding.

Starting with the work of Klein et al. in the early 1950s, there has been a concerted effort to apply the process of freeze-drying for the preservation of platelets in order to provide hemorrhagic patients with a stable infusible hemostatic agent to stop bleeding. The original attempts did not preserve platelet structural integrity and proved to be of little clinical benefit. However, it was known that fixation by various cross-linking agents rendered platelets able to withstand structurally intact the stresses of lyophilization but with (assumed) complete loss of functionality. Read and coworkers showed that fixed and freeze-dried platelets could respond to ristocetin-induced agglutination, and thus devised a widely accepted assay for von Willebrands factor that demonstrated that reconstituted platelets participated well in this in vitro model of an important interaction in primary hemostasis. This review chronicles the efforts of the authors to refine the fixation process so that the freeze-dried and reconstituted platelets retain fundamental hemostatic properties necessary to stop bleeding. The resultant product has demonstrated correction or reduction of the bleeding times in animal models with platelet deficits including the thrombocytopenic rabbit model of Blajchman and coworkers, a canine cardiopulmonary bypass model of open-heart surgery at East Carolina University (ECU), and a porcine trauma model at The University of North Carolina at Chapel Hill (UNC-CH) involving exsanguination and complete blood exchange with a hemoglobin-based oxygen carrier (HBOC). In addition, it has been shown that the fixation process kills viruses and bacteria spiked into the platelet suspension, indicating that the final material may indeed be the first truly sterile cellular transfusion product. The initial goal for clinical benefit is to prevent exsanguination and hypovolemic shock in combat casualties of armed services personnel, for whom platelet transfusions are most often unavailable. Commercial interests are being brought to bear by Entegrion Inc. (formerly known as Hemocellular Therapeutics Corporation) to transfer this technology to a scaleable manufacturing platform for the production of Stasix, a pharmaceutical preparation of fixed and freeze-dried platelets for intravenous or topical use in the arrest of active hemorrhage in a wide variety of patients with a platelet-related bleeding diathesis. It has taken fifty+ years from the first attempt at making a clinically useful freeze-dried platelet preparation to get to the rapidly-approaching clinical trials of Stasix; stabilization of the platelets has been the key to realizing this advance.

Lyophilized canine platelets were infused in a single large bolus dose into splenectomized dogs after 2 hours' perfusion on cardiopulmonary bypass to test their possible efficacy in restoring hemostasis after compromise of platelet function. The vessel bleeding time (VBT) was monitored by venipuncture of the exposed jugular vein. During cardiopulmonary bypass, platelet counts fell quickly and the VBTs became prolonged over baseline. Infusion of lyophilized platelets reconstituted in normal saline occurred just before or immediately after weaning from the cardiopulmonary bypass pump. The results showed consistent and persistent lowering of the VBTs by the infused lyophilized platelets. Controls showed continuously prolonged VBTs. The weighted average VBT in infused subjects was significantly lower than the average in controls: 3 minutes 10 seconds versus 6 minutes 59 seconds, respectively (t test, P = .01). These results in this setting indicate the possible effectiveness of similar human lyophilized platelet preparations in reducing postoperative bleeding in open heart surgery.

BACKGROUND:The rehydrated, lyophilized (RL) platelet (PLT) is being developed as a hemostatic infusion agent for the control of active bleeding. The key to the method for preparing RL PLTs is a mild aldehyde stabilization that allows for freezing and lyophilizing without cellular rupture. RL PLTs have been shown to be effective at rapidly controlling bleeding in animal models of cardiopulmonary bypass induced PLT dysfunction and washout thrombocytopenia, yet the rehydrated cells have proved to be safe with respect to induction of pathologic intravascular coagulation. STUDY DESIGN AND METHODS:In vitro and in vivo studies were performed to better understand the differential effect of the RL PLT manufacturing method on primary and secondary hemostatic processes. The functionality of the von Willebrand factor (VWF) receptor (glycoprotein Ib) complex, the PAR receptors, integrin-mediated aggregation (inside-out signaling), and surface membrane prothrombin to thrombin conversion systems were investigated. RESULTS:RL PLTs were found to retain native VWF-mediated adhesion and surface thrombin generation functions. In contrast, the coupling of thrombin receptors to integrin inside-out signaling was largely inhibited. CONCLUSION:These results suggest that RL PLTs may stop bleeding by forming primary hemostatic plugs and providing a localized source of thrombin for secondary hemostatic processes, yet do not build up occlusive pathologic clots possibly because integrin functions for forming PLT-PLT aggregates are partially inhibited.

Glucosamine- and N-acetyl glucosamine-containing polymers are being used in an increasing number of biomedical applications, including in products for surface (topical) hemostasis. The studies presented here investigate the relationship between the structure (conformation) and function (activation of hemostasis) of glucosamine-based materials. Several polymer systems were studied, including fibers isolated from a microalgal source containing poly-N-acetyl glucosamine polymers that are organized in a parallel, hydrogen-bonded tertiary structure and can be chemically modified to an antiparallel orientation; and gel formulation derivatives of the microalgal fibers consisting of partially deacetylated (F2 gel) and fully deacetylated (F3 gel) polymers. Comparison of the properties of the poly-N-acetyl glucosamine fiber-derived materials with chitin, chitosan, and commercial chitosan-based products are presented. Several studies were performed with the glucosamine-based materials, including (1) an analysis of the ability of materials to activate platelets and turnover of the intrinsic coagulation cascade, (2) an examination of the viscoelastic properties of mixtures of platelet-rich plasma and the glucosamine-based materials via thromboelastography, and (3) scanning electron microscopic studies to examine the morphology of the glucosamine-based materials. The results presented demonstrate that hemostatic responses to the glucosamine-based materials studied are highly dependent on their chemical nature and tertiary/quaternary structure. The unique natural microalgal fibers were found to have strongly prohemostatic activity compared to the other materials studied. (c) 2006 Wiley Periodicals, Inc.

We report on the use of Raman tweezers spectroscopy for the characterization and identification of human platelets for long-term freeze-dried preservation. The paraformaldehyde- fixed, lyophilized and rehydrated platelet samples were spectrally analyzed.

It is a pleasure to find a true compendium on blood platelets that covers the entire range of current knowledge in physiology, pathology, and clinical medicine, and also chronicles the development of each specialty into its own scientific realm. Alan Michelson has assembled an outstanding group of experts to successfully present the breadth of knowledge and advances in our understanding of platelets in bleeding disorders, from basic science to clinical medicine. The foreword, by Barry Coller, gives an excellent history of discoveries in each area of study in platelet research and medicine and, thus, sets the tone for the thoroughness of the chapters that follow.

There is ample evidence for the presence of microvesicles (MV) of different sizes and functions in various blood components. A variety of mechanisms have been proposed for the formation of MV. These include mechanical injury, shear stress, cell activation, activation of complements, hypoxia, and the cell aging process. While MV share many biological properties and surface receptors of their parental cells, they demonstrate significant differences in membrane asymmetry of the inner membrane phospholipid, in particular phosphatidylserine (PS). This provides high-affinity binding sites for the components of the prothrombinase complex. To what extent these MV contribute to hemostatic effectiveness, immudomodulation, and some untoward effects of the transfused blood components remains to be fully elucidated. Several methods for qualitative and semiquantitative characterization of MV are now available. Although in most cases it is necessary to separate MV from the intact cells for improved characterization, recent advances in flow cytometry make it possible to accurately differentiate MV in the presence of their parental cells on the basis of light scattering and fluorescent intensity. This review focuses on four main areas of MV in blood components: (1) the proposed mechanisms of platelet vesiculation, (2) factors influencing the formation of MV, (3) laboratory analysis of MV, and (4) the clinical impact of the presence of MV in blood components. Key Words: Microvesicte—Vesicutation—Biood component—Ptatelets—Transfusion.

: All equipment purchases have been completed. The new Virtus lyophilizer was installed at ECU in August and quickly pressed into service; its first run was on September 15. Now the lyophilizer at the Chapel Hill worksite can be dedicated to bulk animal cell preparations for in vivo testing, while the ECU lyophilizer will be used to optimize preparation technique with human or animal cells. The results of several optimization runs are noted below. The study of metabolic activity of rehydrated platelets was originally proposed as an intramural collaboration at ECU with the Department of Radiation Oncology Biology. Due to a change in faculty, this plan was abandoned and a new arrangement has been made with collaborators at the Mid-Atlantic Regional Headquarters of the American Red Cross to carry out these studies. Stein Holme, Ph.D., established investigator of the American Red Cross and director of the blood research laboratory in Norfolk, VA, will perform oxygen consumption measurements and C(14) adenine radioisotope tracer studies on lyophilized platelet preparations made at ECU or UNC to determine the integrity of metabolic pathways in reconstituted cells. We are optimistic with this new approach because of the past productive collaboration with the Red Cross laboratory during the conduct of our first ONR grant project (N00014-89-J-1712). A subcontract document was signed by both parties September 2, and several samples are now undergoing analysis.