Chitra Krishnamurti

Reduction of blood loss by infusion of human platelets in a rabbit kidney injury model

BACKGROUND: As a first step toward testing the efficacy of stored platelets or platelet substitutes in vivo, a kidney injury model was developed to assess the hemostatic properties of human platelets in normal and thrombocytopenic rabbits.

Survival of fresh human platelets in a rabbit model as traced by flow cytometry

BACKGROUND: In the event of hemorrhage and blood loss, platelets play a vital role in the coagulation process. However, there are currently no acceptable protocols for long‐term storage of platelets. As a first step toward testing the efficacy of stored platelets or platelet substitutes in vivo, a flow cytometric technique was developed to detect human platelets in rabbit blood.

Inhibitory Effects of Lysine Analogues on t-PA Induced Whole Blood Clot Lysis

The lysine analogues epsilon-aminocaproic acid (EACA) and trans-4-amino-methyl cyclohexane carboxylic acid (AMCA) are used to prevent excessive bleeding in patients with coagulopathies, such as hemophilia and thrombocytopenia, or in those who have received tissue plasminogen activator (t-PA). However, their relative efficacy in inhibiting lysis of clots that have been formed in the presence of exogenous t-PA or that have been formed and then exposed to exogenous t-PA has not been well characterized. The present study utilized blood from normal volunteers and 125I-fibrinogen in a dilute whole blood clot assay to determine the relative concentrations of lysine analogues required for inhibition of clot lysis induced by exogenous t-PA. AMCA (0.06 mM) and EACA (0.6 mM) were effective in prolonging clot lysis if (1) whole blood clots were formed and then exposed to a lysine analogue and exogenous t-PA or if (2) whole blood clots were formed in the presence of exogenous t-PA and a lysine analogue. However, their inhibitory effect was markedly reduced if clots were formed in the presence of t-PA and then exposed to either of the lysine analogues. The analogues did not inhibit the initial binding of t-PA to fibrin. They did inhibit binding of plasminogen to fibrin as well as the activation of plasminogen by t-PA in the absence of fibrin. The data suggest that lysine analogues, even at low concentrations, reduce the rate of t-PA induced whole blood clot lysis by several mechanisms.

Cardiovascular toxicity of human cross-linked hemoglobin in a rabbit endotoxemia model.

OBJECTIVE To determine the possible adverse effects of human cross-linked hemoglobin in endotoxemia. DESIGN Prospective, controlled, laboratory trial. SETTING Animal research laboratory. SUBJECTS New Zealand white rabbits. INTERVENTIONS Conscious rabbits received intravenous infusions of either lipopolysaccharide (LPS) alone (10 micrograms/kg, Escherichia coli 0111:B4), human hemoglobin cross-linked between the alpha chains (alpha alpha Hb, 0.7 g/kg), or both LPS and alpha alpha Hb. The cardiovascular effects of alpha alpha Hb and LPS as single agents or administered together were then studied in anesthetized rabbits. MEASUREMENTS AND MAIN RESULTS Mortality in conscious animals that received alpha alpha Hb followed by LPS 4 hrs later (n = 5), or LPS and alpha alpha Hb at the same time (n = 6) was 60% and 67%, respectively. In anesthetized animals, infusion of both LPS and alpha alpha Hb (n = 6) resulted in hypoxia, lactic acidosis, ventricular arrhythmias, and decreased myocardial contractility and left ventricular pressure. In contrast, anesthetized rabbits that received alpha alpha Hb (n = 5) or LPS (n = 5) alone did not develop hypoxia, acidosis, alteration in myocardial contractility, or arrhythmias. Furthermore, death did not occur in any of the conscious animals that received either LPS (n = 7) or alpha alpha Hb (n = 4) as single agents. CONCLUSIONS In an animal model of nonlethal endotoxemia, infusion of alpha alpha Hb significantly increases mortality. Our data suggest that mortality may be due to the acute increased cardiopulmonary toxicity of alpha alpha Hb in animals with underlying endotoxemia.

Stimulated production of urokinase and plasminogen activator inhibitor-2 by the human promyelocytic leukemia cell line HL-60.

The effects of maturation inducing agents on the production of plasminogen activator (PA) and plasminogen activator inhibitor (PAI) by the human promyelocytic leukemia cell line HL-60 were examined. PA activity, which was calibrated with a urokinase standard, was 3-6 mU/10(6) cells when measured in supernatants from control cells. This activity increased at least two-fold after dimethylformamide (DMF) or retinoic acid (RA) was added to cell cultures, and as much as ten to thirty-fold when cells were exposed to 12-O-tetradecanoylphorbol-13-acetate (PMA), an agent that induces monocytoid differentiation in HL-60 cells. The PA activity produced by control and induced cells had the same molecular weight as urokinase (UK), and was completely inhibited by antibodies to UK. Cells that were induced with PMA but not with RA or DMF also produced an inhibitor to UK that was identified as PAI-2, the plasminogen activator inhibitor that is produced by monocytes. Because of its dual capacity to produce both UK and PAI, the HL-60 cell line represents a useful model for studies of the fibrinolytic mediators that are generated and released by leukemia cells.

Addition of a propyl gallate-based procoagulant to a fibrin bandage improves hemostatic performance in a swine arterial bleeding model

Fibrin bandages manufactured by Nycomed Austria (TC-S) were modified by the addition of Hemostyptin (HS), a proprietary platelet-activating reagent containing propyl gallate. HS was added as an additional layer to TC-S fibrin bandages and the bandages were tested for hemostatic efficacy in a swine femoral artery bleeding model. Injuries were treated with a TC-S+HS bandage preparation using HS lyophilized onto a bandage surface that was then attached to the fibrin dressing. This preparation qualitatively and quantitatively exhibited more robust blood clotting at the surgical site than the control bandages. TC-S+HS bandages were more effective than control bandages with a difference in blood loss of 251.8+/-66.5 g for TC-S bandage alone, n=12 vs. 121+/-40.7 g, n=13 for the TC-S+HS bandage, P=0.05. Bleeding times were shortened for animals treated with the HS fortified bandages and residual platelets counts in these animals were higher.

The rabbit as a model for studies of fibrinolysis

When compared to man, the rabbit shows marked prolongation of the dilute whole blood clot lysis time and an attenuated increase in plasminogen activator (PA) after the infusion of desmopressin (DDAVP). The levels of specific components of the plasma fibrinolytic system of the rabbit were compared to those in human plasma to ascertain their role in the differences between species. PA activity and plasminogen levels were similar in the two species. Anti-plasmin and plasminogen activator inhibitor (PAI) activity were lower in the rabbit than in man. The rabbit PAI, apparently similar to that described in man, was not increased by DDAVP infusion. The disparity between man and rabbit with respect to the lysis times of dilute blood clots and response to DDAVP cannot be explained by differences in functional plasma levels of inhibitors or activators of the fibrinolytic system.

Assessment of ω-fatty-acid-supplemented human platelets for potential improvement in long-term storage

Abstract Uptake of omega (ω)−3 fatty acids can influence membrane stability and cell mobility. We investigated the effects of ω−3 and −6 fatty acids on the hemostatic efficacy of human platelets using an in vivo rabbit bleeding model. In vitro assays such as platelet aggregation, vWF bead-mediated ATP release and platelet adhesion to beads (measured by the residual platelet count [RPC] {free platelet count after reacting with the beads}/{baseline platelet count}×100=%RPC; a high %RPC indicates reduced platelet function) were conducted on platelets treated with 1% fish oil (ω−3); 2% fish oil emulsion or 1% soy oil (ω−6). Oil treatment of platelets reduced the vWF bead-induced ATP release insignificantly. Addition of ω−3 agents reduced physical reactivity (%RPC) with the vWF beads by a factor of 1.2 (oil) and 1.9 (emulsion). The ω−6 oil enhanced reactivity by a factor of 1.7. After washing to remove excess reagent, platelet resuspension was most efficient with the ω−3 emulsion. Platelet function was higher with the ω−3-treated platelets (%RPC=52.3%, ω−3 oil; 63.3%, ω−3 emulsion vs. 85%, ω−6 oil; 82% untreated platelets). Ethyl-palmitate-treated thrombocytopenic rabbits were infused with human platelets. Survival times of the treated platelets, as monitored by flow cytometry (6.2–8.2 h) were comparable to untreated platelets (8.6 h). In the rabbit kidney injury model, blood loss after infusion of the treated platelets was similar to that of saline-infused rabbits (75.3±3.4 g). However, platelets washed prior to infusion reduced blood loss to a value comparable to that of fresh platelets (48.3±5 g). Furthermore, the presence of the infused platelets at the injury site was clearly visualized using FITC-tagged anti CD42a antibody. Thus, the ω−3-based agents protect the platelets from damage during the washing procedure as demonstrated in vitro by improved platelet resuspension, low %RPC, high stimulus-responsive ATP secretion and a reduction in blood loss in vivo.

Association of Protein S Deficiency with Thrombosis in a Kindred with Increased Levels of Plasminogen Activator Inhibitor-1

The most common inherited abnormalities associated with familial venous thrombosis are deficiencies of protein S, protein C, and antithrombin III [1-3]. All are inherited in an autosomal dominant manner and can result in venous thrombosis at a young age. The most common of these three deficiencies is protein S, which circulates in a free form and in an inactive complex with C4b-binding protein. Only unbound or free protein S has functional activity as a cofactor for activated protein C in inhibiting factors Va and VIIIa [4]. Thus, assessment of patients for protein S deficiency includes measurement of the level or activity of free protein S. Although assays are now widely available for measuring components of the fibrinolytic system, the importance of decreased fibrinolysis as a risk factor for thrombosis is not well defined [3, 5]. Defective fibrinolysis may be due to increased levels of plasminogen activator inhibitor-1 (PAI-1), a specific inhibitor of tissue-type plasminogen activator. Plasminogen activator inhibitor-1 activity can be increased chronically in some persons such as pregnant women [6], or can undergo acute increases of 3 to 40 times in persons who have had surgery [7] or who are septic [8, 9]. Increased levels of PAI-1 have been reported as a cause of familial thrombosis in one North American kindred [10, 11] and in two European families [12-15]. However, measurements of free protein S were not included in those reports because assays were not yet easily available; further, only symptomatic members were studied. A re-evaluation of the North American kindred was initiated in 1987, because an asymptomatic family member wanted to know his potential risk for thrombosis. The availability of functional assays for the determination of protein C [16] and protein S [17], as well as the continued clinical interest in the role of PAI-1 in promoting thrombosis, prompted more extensive evaluation of this family in 1991 and 1992. Protein S deficiency was documented in 6 of the 7 adults who had a history of venous thromboembolism and in 9 of 21 asymptomatic family members. Plasminogen activator inhibitor-1 activity was increased in some persons; however, PAI-1 activity and a history of thrombosis were not correlated. Our study provides further evidence that the clinical utility of assays for PAI-1 activity in evaluating familial thrombosis has not been established [3, 5]. Methods Patients In 1983, a kindred was reported with venous thrombosis attributed to defective fibrinolysis [10]. In a subsequent study [11], increased plasma PAI-1 activity was measured in a symptomatic person in the kindred, suggesting that increased levels of the inhibitor were the cause of the defective fibrinolysis and thrombosis. In 1987, an asymptomatic, 40-year-old, military family member (who wanted to know his risk factor for thrombosis [III-28]) and his 39-year-old sibling (III-29) (who had a history of venous thrombosis at age 27 years) were evaluated at the Walter Reed Army Institute of Research. Levels of free and total protein S were decreased in the plasma from the sibling with thrombosis; his plasma PAI-1 activity was normal and the tissue plasminogen activator activity was normal before and after venous occlusion [18]. The asymptomatic, active-duty member had normal levels of free and total protein S; however, plasma PAI-1 activity was increased and his tissue plasminogen activator activity was decreased before venous occlusion and did not change after occlusion. These data suggested that protein S deficiency and not increased levels of PAI-1 activity was associated with thrombosis in this family, and further investigation of family members was initiated. All persons gave informed consent under a protocol approved by the Walter Reed Army Institute of Research Human Use Committee. Evaluations were done in the homes of family members or at the Walter Reed Army Institute of Research and occurred at least 4 months after the most recent thromboembolic event. The age, sex, smoking habits, medications, and medical conditions associated with thrombosis were recorded for each participant. Patients were considered smokers if they smoked cigarettes at the time of their previous thrombotic events. At the time of testing, none of the participants was pregnant or had evidence of recent infection or trauma. The date, anatomic location, and treatment of previous thromboembolic events were obtained either from family members, their physicians, or review of medical records. Of the seven patients with a history of venous thromboembolism who participated in this study, thromboses had been documented by ventilation perfusion scans or venography or both in all but one patient (IV-1). The family pedigree (Figure 1) showed a large kindred with a history of thromboembolic events in three generations. Thirty-four adults were interviewed, including all the adult members of the originally reported pedigree (II-7, his spouse II-7s, and their descendants). A total of 28 adults were available for laboratory testing. This included seven surviving persons with a history of thrombosis. In addition, seven asymptomatic children (IV-21, IV-24, IV-25, IV-31, IV-41, IV-42, IV-43) ranging in age from 6 to 17 years were tested for protein S deficiency but were not included in the analysis. Figure 1. Family pedigree. Family History When the family was first described in 1983 [10], five members had a history of venous thromboembolism. Two brothers (III-26 and III-29) had deep venous thrombosis and pulmonary embolism at ages 25 and 27 years, respectively. A third brother (III-25) died at age 29 years from mesenteric thrombosis and pulmonary embolism, which were confirmed at autopsy. Two sons of III-25 had superficial thrombophlebitis (IV-32) and pulmonary embolism (IV-33) at ages 14 and 13 years, respectively. Three years after this initial report, the patriarch of the family (II-7), who was then 64 years old, developed deep venous thrombosis, which was confirmed by ultrasound. He agreed to take warfarin for several weeks only and has not had recurrent thrombosis. He declined to be included in the current family studies. Of the two surviving brothers with a history of thrombosis described in the initial report, one (III-26) has had continued recurrent thrombotic events (deep venous thrombosis, mesenteric vein thrombosis, and pulmonary embolism) at times when he is not receiving warfarin. His brother (III-29) has not had recurrent thrombosis and does not take warfarin. Another family member (IV-32), who is now 29 years old, has not had recurrent thrombosis and usually takes warfarin; his brother (IV-33) died at age 26 years (hospital records not available). All seven previously studied family members who were asymptomatic at the time of the initial report have not had symptoms of venous thromboembolism (III-30, III-31, IV-34 to IV-36, IV-38, IV-41). In addition to the members described in the initial report, our study includes four additional members with a history of thrombosis (III-24, III-22, III-3, and IV-1). Family member III-24 is a 55-year-old man who developed venous thrombosis documented by venogram in his mid-twenties. He subsequently had a pulmonary embolism at age 26 years after vein-stripping surgery. He has since been asymptomatic and does not take warfarin. Formerly a heavy smoker, he stopped smoking cigarettes in his forties. His father (II-6) died in his twenties after complications related to infected leg ulcers and thrombosis sustained after a farming accident. His half-sister (III-24), who is obese and smokes cigarettes, developed deep venous thrombosis and pulmonary embolism on two occasions in her early forties. Each event occurred in the perioperative period, one after a dilatation and curettage and another after hysterectomy. She does not take warfarin. Family member III-3, a 58-year-old nonsmoker, had deep venous thrombosis at ages 45 and 48 years and a pulmonary embolism at age 50. Each of these events occurred when he was not taking warfarin. He continued to take warfarin after his last event until 6 months before this evaluation. His oldest daughter (IV-1) had one episode of leg warmth and swelling at age 31 years, which resolved without anticoagulation medication. At age 36, she was treated with warfarin for 3 months for the same symptoms without having diagnostic testing. One month after discontinuation of warfarin she developed pleuritic chest pain, hemoptysis, and shortness of breath. She was empirically treated with antibiotics and her symptoms resolved gradually over several weeks. She does not take warfarin. Assays Blood Collection No participants were anticoagulated with warfarin or with heparin at the time of these studies. All blood samples were obtained between the hours of 8:00 a.m. and 10:00 a.m. from persons who had fasted for 12 hours and who had abstained from smoking and from any form of exertion for at least 1 hour before phlebotomy. Blood was drawn by venipuncture through a 21-gauge butterfly needle, was mixed with 3.8% (weight/volume) trisodium citrate anticoagulant (9 parts blood:1 part anticoagulant) in a polypropylene tube, and was immediately centrifuged at 12 500 x rpm in an Eppendorf microfuge 3200 (Brinkman Instruments, Westbury, New York) for 3 minutes at 22 C. The separated plasma was immediately aliquoted and frozen at 40C. Protein S Assays Functional protein S activity was determined by measuring its cofactor activity for activated protein C in a modified, activated partial thromboplastin time assay (Staclot Protein S, American Bioproducts, Parsippany, New Jersey). A laboratory reference range (mean 2 SD) was determined in 23 healthy men and 22 healthy women who were not pregnant or receiving oral contraceptives. The values were expressed as the percentage of protein S activity in pooled plasma obtained from 18 normal volunteers (9 men, 9 women; 34 13 years [mean SD]). An enzyme-linked immunosorbent assay p

Biological Consequences of Cross-linked Hemoglobin in Animal Models of Surgery and Endotoxemia

Cross-linked purified human hemoglobin produced by several companies has been administered successfully to multiple individuals in phase I clinical trials and is now undergoing evaluation in phase II studies. Since hemoglobin will be administered in acute care settings to patients who may have limited cardiovascular reserve or who are septic, its potential biologic activities other than the ability to carry oxygen require consideration. Hemoglobin has an affinity for nitric oxide (NO), a gas with multiple physiologic functions, that is orders of magnitude greater than for oxygen. NO has a dominant role as a vasodilator in regulating blood pressure, as well as in inhibiting adhesion and aggregation of platelets and leukocytes (Moncada and Higgs 1993).