Blockade of C5a and C5b-9 generation inhibits leukocyte and platelet activation during extracorporeal circulation.

Complement activation contributes to the systemic inflammatory response induced by cardiopulmonary bypass. At the cellular level, cardiopulmonary bypass activates leukocytes and platelets; however the contribution of early (3a) versus late (C5a, soluble C5b-9) complement components to this activation is unclear. We used a model of simulated extracorporeal circulation that activates complement (C3a, C5a, and C5b-9 formation), platelets (increased percentages of P-selectin-positive platelets and leukocyte-platelet conjugates), and neutrophils (upregulated CD11b expression). to specifically target complement activation in this model, we added a blocking mAb directed at the human C5 complement component and assessed its effect on complement and cellular activation. Compared with a control mAB, the anti-human C5 mAb profoundly inhibited C5a and soluble C5b-9 generation and serum complement hemolytic activity but had no effect on C3a generation. Additionally, the anti-human C5 mAb significantly inhibited neutrophil CD11b upregulation and abolished the increase in P-selectin-positive platelets and leukocyte-platelet conjugate formation compared to experiments performed with the control mAb. This suggests that the terminal components C5a and C5b-9, but not C3a, directly contribute to platelet and neutrophil activation during extracorporeal circulation. Furthermore, these data identify the C5 component as a site for therapeutic intervention in cardiopulmonary bypass.

Role of C3 cleavage in monocyte activation during extracorporeal circulation.

BACKGROUND: We previously demonstrated that inhibiting formation of terminal complement components (C5a and C5b-9) prevents platelet and neutrophil (PMN) but not monocyte activation during simulated extracorporeal circulation (SECC). This study examined whether earlier complement inhibition during SECC, blocking C3a formation, would additionally prevent monocyte activation. METHODS AND RESULTS: SECC was established by recirculating heparinized whole blood from human volunteers on a membrane oxygenator. CAB-2, a chimeric protein constructed from genes encoding the complement regulatory proteins CD46 and CD55, inactivates the C3/C5 convertases and blocks in vitro generation of C3a, C5a, and C5b-9. CAB-2 was used in 4 experiments at a final concentration of 300 micrograms/mL and 4 experiments at 30 micrograms/mL; 4 control runs used vehicle alone. Samples were assayed for C3a and C5b-9, monocyte activation (CD11b upregulation), PMN activation (CD11b upregulation and elastase release), and platelet activation (P-selectin expression and monocyte-platelet conjugate formation). CAB-2 at both doses significantly inhibited formation of C3a and C5b-9 during SECC. High-dose CAB-2 significantly blocked monocyte and PMN CD11b upregulation and PMN elastase release. CAB-2 also inhibited formation of platelet activation-dependent monocyte-platelet conjugates. CONCLUSIONS: Blockade of complement activation early in the common pathway inhibited monocyte CD11b upregulation during SECC, suggesting that early complement components contribute most to monocyte activation during SECC. As expected, PMN and platelet activation were blocked by terminal complement inhibition. This investigation further elucidates the relation between complement and blood cell activation during simulated cardiopulmonary bypass.

Characterization of the P-selectin ligand on human hematopoietic progenitors.

The adhesive characteristics of hematopoietic stem and progenitor cells may partly regulate their proliferation and differentiation and may be critical in the homing of transplanted stem cells. Using quantitative adhesion assays, we have examined the characteristics of activated platelet adhesion to CD34+ cells in human blood and to the KG1a cell line. Approximately 85-95% of CD34+ cells from both sources bound thrombin-activated platelets; like mature neutrophils, activated platelet binding was maximal within 2 minutes of coincubation. Activated platelet adhesion to CD34+ cells was completely inhibited by chelation of calcium or by preincubation with the G1 blocking monoclonal antibody (MoAb) to platelet P-selectin. Using MoAbs to P-selectin glycoprotein ligand-1 (PSGL-1), we demonstrated that PSGL-1 was present on the surface of CD34+ cells; preincubation of CD34+ cells with the PL1 blocking MoAb to PSGL-1 completely inhibited activated platelet adhesion to CD34+ cells. Furthermore, treatment of CD34+ cells with O-sialoglycoprotein endopeptidase, which destroyed the PL1 epitope of PSGL-1, also abolished activated platelet-CD34+ cell binding. By contrast, MoAb directed against control epitopes of PSGL-1 or endopeptidase-sensitive epitopes of the CD34 molecule had no effect on activated platelet adhesion to CD34+ cells. Unlike mature neutrophils that, when activated, decrease P-selectin-dependent platelet adhesion because of redistribution of PSGL-1, phorbol ester treatment of CD34+ cells had no effect on their ability to bind activated platelets or PSGL-1 MoAbs. This study identifies PSGL-1 on CD34+ cells as the ligand for platelet P-selectin and suggests functional differences between mature and precursor hematopoietic cells in the regulation of surface PSGL-1 expression.

Interactions of platelets and endothelial cells with erythrocytes and leukocytes in thrombotic thrombocytopenic purpura.

In both normal and abnormal vascular biology, platelet-leukocyte-RBC-EC adhesive and functional interactions appear to play a significant role. The clinical hallmarks of the TTP/HUS syndromes, including prominent platelet aggregation and thrombi without prominent soluble coagulation system activation along with microangiopathic hemolysis, suggest that such interactions may help to bring a common theme of explanation to this disorder. However, a final unifying physiological pathway remains elusive.

Neutrophil but not monocyte activation inhibits P-selectin-mediated platelet adhesion.

Selectins are Ca(2+)-dependent glycoprotein receptors that mediate the adhesion of activated platelets or endothelial cells to unstimulated leukocytes. Using purified cell fractions, we examined activated neutrophil adhesion to P-selectin-expressing platelets and found that phorbol 12-myristate 13-acetate (PMA), platelet activating factor C16 (PAF), and n-formyl-met-leu-phe (fMLP) pretreatment of neutrophils inhibited activated platelet adhesion. Furthermore, PMA and PAF were capable of dissociating established resting neutrophil-activated platelet conjugates. Since L-selectin is downregulated after leukocyte activation and has been postulated as a ligand for P-selectin, we preincubated resting neutrophils with Dreg-2 and Dreg-56, blocking monoclonal antibodies (MoAb) to L-selectin; these MoAb failed to inhibit activated platelet adhesion. To more closely approximate in vivo conditions of leukocyte and platelet activation, we also employed a whole blood (WB) model of leukocyte-platelet adhesion. We found that simultaneous activation of both platelets and leukocytes by PMA caused an immediate rise in the % of P-selectin-positive platelets accompanied by a rapid increase in monocyte-platelet and neutrophil-platelet conjugates; however, the % of neutrophil-platelet conjugates subsequently declined over 30-60 min to baseline levels while monocyte-platelet adhesion remained elevated over 90 min. By contrast, selective platelet activation in WB by thrombin resulted in an increase in platelet P-selectin expression accompanied by a sustained (90 min) elevation in both monocyte- and neutrophil-platelet conjugates. This increase in leukocyte-platelet conjugates after thrombin was not inhibited by preincubation of WB with Dreg-2 or Dreg-56. We conclude that neutrophil activation decreases the expression of the ligand for platelet P-selectin within 30-60 min resulting in inhibition of neutrophil-platelet adhesion and dissociation of existing neutrophil-platelet conjugates.(ABSTRACT TRUNCATED AT 250 WORDS)

Differences in platelet alpha-granule release between normals and immune thrombocytopenic patients and between young and old platelets.

The risk of serious bleeding in patients with immune thrombocytopenic purpura (ITP) appears to be less than in comparably thrombocytopenic patients with megakaryocytic hypoplasia. It has been proposed that this difference is due to enhanced hemostatic activity of young platelets, which are increased in the circulation during ITP. We examined alpha-granule release in reticulated platelets (RP), which are thought to be the youngest circulating platelets, and in older non-reticulated platelets (non-RP) in normal human controls and ITP patients. Normal controls had a mean RP of 7%, compared with 42% in ITP patients. The mean concentration of thrombin receptor agonist peptide (TRAP) causing 50% of control RP to express CD62P (EC50) was 0.82+/-0.08 microM (SEM), significantly higher than the TRAP CD62P EC50 for RP in ITP, 0.57+/-0.06 microM (p = 0.04). Similarly, the TRAP EC50 for non-RP in controls, 0.84+/-0.09 microM, was significantly higher than in ITP, 0.56+/-0.07 microM (p = 0.03), suggesting that all platelets in ITP have an enhanced alpha-granule threshold response to TRAP compared with controls, while RP and older platelets within each patient group have similar threshold sensitivities to TRAP. By contrast, high-dose TRAP caused RP to express twice as much mean and total CD62P as non-RP in both ITP patients and controls (p <0.05 for both comparisons). We conclude that compared with controls, all platelets in ITP are primed to undergo alpha-granule release to TRAP, while in both ITP and controls, the newly circulating, reticulated platelets have the potential to contribute greater amounts of CD62P surface ligand compared with older platelets (non-RP) after stimulation. Both the increased RP% and enhanced platelet response to agonist in ITP may contribute to maintenance of hemostasis despite thrombocytopenia.

Selective blockade of membrane attack complex formation during simulated extracorporeal circulation inhibits platelet but not leukocyte activation.

OBJECTIVE: Complement activation is induced by cardiopulmonary bypass, and previous work found that late complement components (C5a, C5b-9) contribute to neutrophil and platelet activation during bypass. In the present study, we blocked C5b-9 formation during extracorporeal recirculation of whole blood to assess whether the membrane attack complex was responsible for both platelet and leukocyte activation. METHODS: In a simulated extracorporeal model that activates complement (C3a and sC5b-9), platelets (CD62P expression, leukocyte-platelet conjugate formation), and leukocytes (increased CD11b expression and neutrophil elastase), we examined an anti-human C8 monoclonal antibody that inhibits C5b-9 generation for its effects on cellular activation. RESULTS: Anti-C8 significantly inhibited sC5b-9 formation but did not block C3a generation. Anti-C8 also significantly inhibited the increase in platelet CD62P and monocyte-platelet conjugate formation seen with control circulation. Moreover, compared with control circulation, in which the number of circulating platelets fell by 45%, addition of anti-C8 completely preserved platelet counts. In contrast to blockade of both C5a and sC5b-9 during simulated extracorporeal circulation, neutrophil activation was not inhibited by anti-C8. However, circulating neutrophil and monocyte counts were preserved by addition of anti-C8 to the extracorporeal circuit. CONCLUSIONS: The membrane attack complex, C5b-9, is the major complement determinant of platelet activation during extracorporeal circulation, whereas C5b-9 blockade has little effect on neutrophil activation. These data also suggest a role for platelet activation or C5b-9 (or both) in the loss of monocytes and neutrophils to the extracorporeal circuit.

In vitro and in vivo evaluation of platelet transfusions administered through an electromechanical infusion pump.

The authors evaluated two platelet infusion pump systems, the Abbott Lifecare 5000 and the Omniflow 4000, for evidence of in vitro platelet damage and in vivo platelet recovery. When compared with gravity infusion, there was no significant difference in levels of LDH discharge, beta-thromboglobulin release, cell counts, or morphology score after platelet concentrates were infused through these pumps. When the pumps were compared to gravity infusion in thrombocytopenic oncology patients, no differences were noted in platelet-corrected count increments at 1-4 hours or 12-24 hours post-transfusion. The authors conclude that these infusion systems do not significantly injure or activate platelets and that they are efficacious for transfusing platelet concentrates to thrombocytopenic patients. These infusion pumps may be of clinical benefit to pediatric or adult patients with a history of prior transfusion reactions, when precise control of the rate and volume of platelet transfusion is desired.

The significance of platelets with increased RNA content (reticulated platelets). A measure of the rate of thrombopoiesis.

Direct flow cytometric measurement of nucleic acid content in individual platelets is possible using the fluorescent dye Thiazole Orange (Becton-Dickinson, San Jose, CA). When applied to studies of thrombocytopenic patients, platelets with elevated nucleic acid content ("reticulated platelets") can be identified and quantitated. Labeling of these platelets is saturable and is abolished by treatment with RNAse. It has been suggested that, similar to the erythrocyte reticulocyte response to anemia, the number of these platelets appearing in the circulation may provide an estimate of the rate of thrombopoiesis. The authors studied 229 thrombocytopenic patients, measuring both reticulated platelets and platelet-associated immunoglobulin. The results show that for the subset of patients with normal levels of platelet-associated immunoglobulin, the average absolute number of reticulated platelets is independent of platelet count and remains in the normal range. For those with elevated levels of platelet-associated immunoglobulin, the absolute number of reticulated platelets increases in patients who are moderately thrombocytopenic (60 to 100 x 10(9)/L) but decreases to normal or subnormal levels as thrombocytopenia worsens. The latter finding has been duplicated in studies of mice made thrombocytopenic by injection of anti-platelet antiserum. These results are consistent with the hypothesis that reticulated platelets are subject to peripheral destruction at the same rate as mature platelets, and that in the severely thrombocytopenic patient their level may decrease despite an appropriate marrow thrombopoietic response.

Cocaine, HIV, and their cardiovascular effects: is there a role for ACE-inhibitor therapy?

Cocaine abuse and HIV disease each have potentially adverse effects upon the heart and cardiovascular system which may be exacerbated when these risk factors are combined. The development of a safe and effective agent to treat both cocaine addiction and its cardiovascular sequelae, that is well-tolerated by HIV patients, would thus be of considerable clinical utility. In this article we discuss the rationale for the investigation of angiotensin converting enzyme (ACE) inhibitors, commonly used to treat hypertension, for treatment in cocaine-abusing populations, based on their potential to reduce cocaine use by modulating levels of dopamine and corticotropin releasing factor in the brain, and on their ability to reverse cardiovascular and platelet abnormalities. We present preliminary findings from echocardiographic and platelet activation studies in 16 HIV-positive, cocaine abusing patients, as well as tolerability and efficacy studies of the ACE-inhibitor, fosinopril, for the treatment of cocaine abuse in both HIV-positive (n=6) and HIV-negative (n=5) methadone-maintained cocaine abusers. Findings suggest that HIV-positive cocaine-abusing patients possess abnormalities of diastolic heart function and platelet activation that are potentially reversible with ACE-inhibitor therapy. Findings also suggest that fosinopril is well-tolerated regardless of HIV serostatus, does not appear to cause hypotension, and may possess effectiveness for reducing cocaine use. We conclude that ACE-inhibitor therapy may offer a new pharmacologic approach to the treatment of cocaine abuse and its complications, and that controlled research of this class of agents may be promising.

Stroke in a neonate heterozygous for factor V Leiden.

Activated protein C resistance is the most common hereditary coagulation abnormality and is caused by the factor V Leiden mutation. A newborn who developed seizures within hours after delivery and was found to have a bihemispheric stroke is described. This patient, determined to be heterozygous for factor V Leiden, is the first reported case of neonatal stroke associated with this common mutation.

Reticulated platelets in the evaluation of thrombopoietic disorders.

The laboratory diagnosis of immune platelet destruction has relied predominantly on the presence or absence of megakaryocytes in bone marrow. Recently, examination of peripheral blood platelets for high RNA content (reticulated platelets) or for elevated levels of platelet-associated IgG have been suggested as less invasive diagnostic tests. We used thiazole orange fluorescence labeling to determine the percentage of circulating reticulated platelets and two antibodies with different specificities directed against human IgG to measure platelet-associated IgG by flow cytometry in 59 patients with either immune thrombocytopenic purpura (n = 23) or chemotherapy-induced thrombocytopenia (n = 36). The percentage of reticulated platelets in patients with immune thrombocytopenia was significantly increased (38.6% +/- 27.4% [mean +/- 1 SD]), compared with patients receiving chemotherapy and normal subjects (7.2% +/- 3.3% and 2.9% +/- 2.2%, respectively). However, 17% of patients with immune thrombocytopenia had reticulated platelet values in the range observed for normal subjects and for patients with chemotherapy. Although one third of patients with immune thrombocytopenia had very high platelet IgG levels, the majority could not be distinguished from patients receiving chemotherapy solely on this basis. Combining the reticulated platelet determination with the IgG data did not improve the sensitivity or specificity of the reticulated platelet determination alone. We conclude that a flow cytometric assay for reticulated platelets is a better discriminant than flow-measured platelet IgG for diagnosing immune platelet destruction. We further postulate that the subset (17%) of patients with immune destruction who have relatively low percentages of reticulated platelets may represent patients with an inappropriately low thrombopoietic response.

Platelet function testing by flow cytometry.

The availability of fluorescent monoclonal antibodies and probes now provides a powerful tool for examining platelet function by flow cytometry. These techniques cna be employed to determine the resting and stimulated expression of platelet glycoprotein receptors, the activation status of platelets assessed by secretion of granule contents (including expression of activation-dependent neoantigens), adhesion ligand binding to platelets, and intracellular calcium flux after exposure of platelets to agonist. In addition, flow cytometry has now been used to study the functional properties of stimulated platelets, including microparticle generation, platelet aggregation, and platelet-heterotypic cell conjugate formation. This brief review is presented as a general outline of the literature that uses flow cytometric methodology to examine in vivo and ex vivo platelet function.

WAG-F8(m1Ycb) rats harboring a factor VIII gene mutation provide a new animal model for hemophilia A.

BACKGROUND: We recently described an inherited coagulopathy arising in an inbred colony of WAG/RijYcb rats. The bleeding phenotype, demonstrated by both male and female rats, included periarticular hemorrhage, spontaneous bruising, prolonged bleeding from minor wounds and maternal peripartum deaths. Coagulation testing of affected rats revealed normal prothrombin time but prolongation of activated partial thromboplastin time to twice that of controls. OBJECTIVE: To determine the specific coagulation factor and the underlying genetic defect responsible for the inherited coagulopathy in the WAG/RijYcb rats. RESULTS: Evaluation of individual clotting factor activities revealed that the affected animals had a specific deficiency of factor (F) VIII (FVIII). The FVIII gene (F8) has an autosomal location on chromosome 18 in rats, in contrast to its location on the X chromosome in mice and humans. Sequencing of F8 cDNA led to the identification of a point mutation resulting in a substitution, Leu176Pro, in the A1 domain, that is predicted to disrupt the tertiary structure of the FVIII molecule. Administration of human plasma or human recombinant FVIII corrects the coagulation abnormality in the affected animals. CONCLUSIONS: We have now identified the genetic basis of the hemostatic defect in the WAG/RijYcb rat colony. The larger size of rats relative to mice and the presence of this coagulation defect in both sexes provide a unique model, well-suited to the development of novel therapies for acquired and hereditary FVIII deficiencies.

Activation of platelet concentrate during preparation and storage.

This review will discuss how stored platelets become activated and will examine their ability to function and survive in vivo, posttransfusion. Experimental methods which have been shown to alter platelets during storage will be detailed. Using beta-thromboglobulin (beta-TG) and surface adhesion receptors as markers, investigators have examined the activation changes in platelet concentrates during preparation and storage. Resuspension of the platelet pellet after isolation of platelet-rich plasma appears to play a major role in producing platelet activation and beta-TG release during preparation. However, there is a significant amount of interdonor variability in platelet activation even at this early stage of storage. Over 5 days of storage, platelets release approximately 50% of their beta-TG contents. Furthermore, between 40% and 60% of the platelets express the alpha-granule membrane protein, P-selectin (GMP-140), during storage, which is also indicative of platelet activation. These activation changes correlate to some degree with platelet recovery posttransfusion but clearly do not explain the full lesion of platelet storage. The surface density of two platelet membrane receptors, glycoproteins (GP) Ib and IIb/IIIa, also change with activation, although in opposite directions. Platelet surface GPIb decreases initially with storage and then recovers, perhaps due to its relocation to the platelet surface from an intracellular pool. In contrast to GPIb, mean platelet surface GPIIb/IIIa increases slightly during storage, probably as a consequence of platelet activation and release of alpha-granule GPIIb/IIIa to the surface. Some hypotheses are offered regarding how these activated platelets can continue to circulate after transfusion. Further exploration of the platelet storage lesion will hopefully provide needed answers and thus permit better treatment of hemostatic disorders in the future.

Impact of unusual gastrointestinal problems on the treatment of tricyclic antidepressant overdose.

We report the cases of two patients with tricyclic antidepressant overdose in which the use of charcoal was hampered by gastrointestinal abnormalities. In the first patient, a previous gastric bypass procedure impeded the placement of an orogastric tube and subsequent charcoal administration, while potentially facilitating rapid absorption of the drug--factors that may have contributed to her death. In the second patient, severe esophageal spasm delayed therapy until IV nitroglycerin relieved the functional obstruction. Both circumstances are previously unreported complications associated with tricyclic antidepressant overdose. Potentially corrective measures are proposed.

Septic anaerobic jugular phlebitis with pulmonary embolism: problems in management.

Anaerobic gram-negative bacillary bacteremia and multiple septic pulmonary emboli developed rapidly in two previously healthy young men after an episode of pharyngitis. One patient developed proptosis and subsequent uniocular blindness. In both cases facial swelling was an early sign of jugular vein involvement. In patients not responding to antibiotic therapy, systemic anticoagulation or surgical venous ligation may be potentially useful as an additional therapeutic measure. Septic jugular vein phlebitis is a serious condition that requires early recognition and rapid institution of appropriate therapy.