Heparin-induced thrombocytopenia is characterized by moderate thrombocytopenia and thrombotic complications, whereas quinine/quinidine-induced thrombocytopenia usually presents with severe thrombocytopenia and bleeding. Using flow cytometry and assays of procoagulant activity, we investigated whether sera from patients with these immune drug reactions could stimulate normal platelets to generate platelet-derived microparticles with procoagulant activity. Sera or purified IgG from patients with heparin-induced thrombocytopenia stimulated the formation of platelet-derived microparticles in a heparin-dependent fashion. Further studies showed that heparin-induced thrombocytopenia sera also produced a marked increase in procoagulant activity. In contrast, sera from patients with quinine- or quinidine-induced thrombocytopenia did not generate platelet-derived microparticles nor generate increased procoagulant activity. However, quinine/quinidine-induced thrombocytopenia sera produced a significant increase in the binding of IgG to platelets in a drug-dependent fashion, whereas sera from patients with heparin-induced thrombocytopenia demonstrated no drug-dependent binding of IgG to platelets. We also observed increased levels of circulating microparticles in patients with acute heparin-induced thrombocytopenia compared with control patients. Our observations indicate that the generation of procoagulant platelet-derived microparticles in vivo is a plausible explanation for the thrombotic complications observed in some patients with heparin-induced thrombocytopenia.

Currently, therapeutic platelet concentrates can be stored for only 5 days. We have developed a procedure that permits long-term storage of fixed and lyophilized platelets that retain hemostatic properties after rehydration. These rehydrated lyophilized platelets (RL platelets) restore hemostasis in thrombocytopenic rats and become incorporated in the hemostatic plug of bleeding time wounds of normal dogs as well as von Willebrand disease dogs with partially replenished plasma von Willebrand factor. Ultrastructurally, these platelets are well preserved and are comparable to control normal washed platelets. Flow cytometry analysis shows that RL platelets react with antibodies to the major surface receptors, glycoprotein (GP)Ib and GPIIb/IIIa. These receptors are involved in platelet agglutination, aggregation, and adhesion. In vitro functional tests document the ability of RL platelets to adhere to denuded subendothelium and to spread on a foreign surface. Circulating RL platelets participated in carotid arterial thrombus formation induced in normal canine subjects. The participation of RL platelets in these vital hemostatic properties suggests that with further development they could become a stable platelet product for transfusion.To promote effective hemostasis, platelets must respond quickly to changes in normal blood flow or vessel injury (1, 2). After vascular injury, platelets adhere to exposed subendothelium, aggregate, and form a primary platelet plug. Platelet activation and initiation of coagulation follow with stabilization of the platelet plug by the formation of fibrin. The initiation of a thrombus at a site of vascular injury is mediated through platelet membrane glycoprotein (GP) receptors (3, 4). Platelet adhesion to a damaged vessel wall and its extracellular matrix at high shear is primarily mediated through the specific interaction of the platelet membrane GPIb-IX complex and bound von Willebrand factor (vWF) (5-7), which is synthesized and released into plasma and the vessel wall by endothelial cells (1). Platelet adhesion at low shear rates is mediated by several interactions, including collagen with the a2131 integrin (7). Platelet adhesion stimulates a spreading of the platelet (8).Although the mechanism of platelet spreading has not been completely characterized, recent in vitro studies have shown that platelets will spread on surfaces coated with fibrinogen (9) or polymerized fibrin (10). The activation of the GPIIb/IIIa receptor by agents such as ADP results in a conformational change in the receptor (11-13). The activated receptor binds fibrinogen, which forms a "bridge" between the platelets, and causes aggregation (1,14,15). Activated platelets provide the phospholipid surface for the assembly of blood clotting enzyme complexes, and the concentration and localization of activated coagulant proteins at sites of vessel wall injury may be facilitated by adherent platelets (16). Internal storage granules in platelets release clot-promoting contents in response to activation of bi...

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 × 109/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 LYOor 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 LYOand 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. LYOwere easy to use and provided storage advantages over FRESH. Further study of this product, including examination of efficacy and platelet life span, is warranted.

No abstract

Membranous microparticles (MP) appearing in the supernatant plasma of stored platelet concentrates (PC) were analyzed by flow cytometry. Two populations of MP were arbitrarily delineated by light scatter as larger or smaller than 0.5 micron fluorescent beads. An estimate of MP concentration was obtained by adding a known amount of fluorescent beads to each sample before analysis of a set number of counts on the flow cytometer. The addition of platelet activation inhibitors (prostaglandin E-1, theophylline, and aprotinin) to the anticoagulant during preparation of PC combined with a reduction in surface area of the storage container caused approximately a 40% reduction in the number of MP appearing during storage relative to donor-matched controls. In addition, the inhibited concentrates had 84% less platelet factor 3 (PF3) activity in the supernatant and 61% less released lactic dehydrogenase. A reduction in surface area of the container in the controls partially offset these differences. A significant correlation was found (rs = .748) between PF3 levels and the concentration of larger MP. The inhibitors did not reduce the small number of MP found in stored platelet-poor plasma. Surface antigen analysis showed that the majority of MP in PC were platelet-derived; most were positive for glycoprotein (GP) IIbIIIa (73%) and/or for GPIb (43% to 46%). We conclude that procoagulant MP are released from platelets during storage as a result of platelet activation augmented by interaction of platelets with the bag wall.

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.

These data suggest the equivalence of FFP and SDP on modulation of endothelial function and inflammation in vitro.

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.