Antithrombotic action of annexin V proved as efficient as direct inhibition of tissue factor or thrombin.

The role of phospholipid platelet membrane and tissue factor in thrombin generation and thrombus formation is accepted. In the present study we have explored antithrombotic action of strategies aimed to block exposure of negatively charged phospholipids and we compared effects with those obtained through tissue factor or a direct thrombin inhibition. Type III collagen was exposed to flowing blood (5 min, 300 s(-1)). Effects of inhibition of platelet deposition by annexin A5 (ANXA5), hirudin (HIR) or by an antibody against tissue factor (TF) were evaluated. Prothrombin fragment F1 + 2 (F1 + 2) was monitored. Pre-incubation of whole blood with HIR or ANXA5 resulted in a statistically significant reduction of platelet deposition (12.2 +/- 0.6% in control experiments vs. 8.3 +/- 0.4% and 8.5 +/- 0.5%, respectively, P < 0.05). A similar decrease was found when blood was incubated with an antibody against TF. Furthermore, ANXA5 and HIR inhibited the recruitment of platelets into forming aggregates. The height of platelet aggregates generated was decreased in the presence of HIR or ANXA5, but only incubation with both inhibitors reached levels of statistical significance. The presence of ANXA5 or HIR decreased levels of F1 + 2 suggesting a reduced activation of the coagulation system. In our experimental studies, the inhibitory potential of ANXA5 on platelet-thrombus formation was as effective as that of a direct thrombin inhibitor, as HIR, or an antibody against TF. Negatively charged phospholipids exposed on activated platelets potentiate the formation of platelet aggregates on a collagen surface and further suggest that inhibition of platelet procoagulant activity might be a specific target for antithrombotic drugs.

In vitro evaluation of platelet reactivity toward annuloplasty devices treated with heparin coating: studies under flow conditions.

We have applied an in vitro perfusion model to explore the potential thrombogenicity of polyester annulolasty fabric used in valve repair and to investigate the possible thromboresistance characteristics conferred by a special heparin coating (Duraflotrade mark treatment). Samples of human blood from i) untreated or ii) heparin-coated extracorporeal circuits were recirculated through annular perfusion chambers containing a) untreated or b) treated annuloplasty cloth material. Perfusion experiments were performed at a shear rate of 600 s(-1) for 20 min. Platelet interaction with the material was morphometrically evaluated. In experiments performed with blood from untreated circuits and cloth material, the average cross-sectional area of platelet mass was 615 +/- 135 microm2. Treatment of cloth material with Duraflotrade mark statistically decreased the area of interacting platelets to 319 +/- 101 microm2 (*p < 0.05, n = 10). Blood samples from heparin-coated extracorporeal circuits showed a decrease of total area of platelets (308 +/- 58 microm2 vs 138 +/- 30 microm2, *p < 0.05, n = 9). The combined treatment of Duraflotrade mark in extracorporeal circuits and cloth material caused a more consistent reduction (p < 0.05). The in vitro perfusion experimental model was sensitive to evaluate the thrombogenic potential of Duraflotrade mark treatment. Our results indicate that the heparin coating of cloth material and extracorporeal circuits improves the biocompatibility of the original material and reduces the thrombogenic profile.

Differences and similarities in tyrosine phosphorylation of proteins in platelets from human and pig species.

BACKGROUND: Pigs have been widely used as animal models to study hemostasis. However, there are significant differences when comparing the hemostatic behavior of pig and human platelets. OBJECTIVE: To investigate signaling through tyrosine-phosphorylation of proteins in pig platelets after activation in suspension or by adhesion under flow conditions, in comparison with human platelets. METHODS: Activation of platelet suspensions was performed with thrombin (T; 0.1 and 1 U mL(-1)) and type I collagen (Col-I; 20 microg mL(-1)), at two different time points (30 and 90 s). Activation by adhesion was carried out on Col-I-coated coverslips, using citrated whole blood samples perfused through a parallel-plate chamber. RESULTS AND CONCLUSIONS: Significant differences between pig and human platelets were detected before and after activation. Activation of pig platelets required higher concentrations of thrombin, as well as increased activation times, to achieve similar levels of tyrosine phosphorylation. Proteins p160, p140, p85 and pp62, present in human platelets, were not detected in profiles corresponding to activated pig platelets. A protein of 70 kDa appeared only in pig platelet profiles, p55 was highly phosphorylated, and the phosphorylation levels of some proteins were significantly different from those found in human platelet profiles. In profiles corresponding to adhered pig platelets, p85 and p62 were absent, and p115 appeared highly phosphorylated. As observed in suspension studies, p70 and p55 appeared specifically in adhered pig platelets. Our study shows that the phosphotyrosine proteins involved in the activation of pig platelets are significantly different from those observed in activated human platelets. These findings may help to explain the differing adhesive and cohesive properties of platelets from both species, which should be considered when extrapolating results.

Clinical significance of acquired activated protein C resistance in patients with systemic lupus erythematosus.

Antiphospholipid antibodies (aPL) may induce acquired activated protein C resistance (acquired APCR). The role of acquired APCR in patients with systemic lupus erythematosus (SLE) is not well known. To evaluate the prevalence of acquired APCR and its association with clinical manifestations we studied 103 consecutive SLE patients and 103 matched controls. APCR in the undiluted test and after dilution in factor V deficient plasma, factor V Leiden, protein C and S, lupus anticoagulant, and anti-cardiolipin, anti-beta2-glycoprotein I and anti-prothrombin antibodies were determined. Factor V Leiden was found in 4% in both patients and controls. The prevalence of acquired APCR was 22% for the undiluted assay and 17% in the diluted test. In SLE patients, acquired APCR was associated with aPL (39 vs 13% in undiluted assay, P = 0.007; and 33 vs 7% in the diluted test, P = 0.001). Arterial thromboses were found in 24% of patients with acquired APCR and in 6% of patients without (P = 0.04). However, no relationship was found with venous thrombosis. Acquired APCR was also associated with pregnancy losses: miscarriages in 70% of women with acquired APCR vs 32% in those without (P=0.03). Thus, in SLE patients acquired APCR seems to be associated with increased prevalence of arterial thrombosis and pregnancy losses.

[Uremic media affects hemostatic properties of human endothelial cells in culture and increases the production of von Willebrand factor]. / El medio urémico altera las propiedades hemostáticas de células endoteliales humanas en cultivo y aumenta la producción de factor von Willebrand.

We have investigated the ability of serum from uremic patients to modify the thrombogenic properties of the endothelium. The effect of the uremic media on the morphology of ECs, and their resistance to flow was analyzed. The reactivity of the extracellular matrix (ECM) generated by ECs towards normal platelets was evaluated in a parallel-plate perfusion chamber. Exposure of ECs to uremic media resulted in abnormal morphology and signs of accelerated growth. Detachment of ECs exposed to circulating blood was increased when cells had been grown with media supplemented with uremic serum (22% vs 13%). Platelet deposition and formation of aggregates were significantly elevated on ECMs generated in the presence of uremic media (40.23 +/- 6.43% vs 25.42 +/- 2.69%, p < 0.05, n = 5). Immunocytochemical methods detected an enhanced expression of von Willebrand factor antigen on uremic ECMs (uremic 17.1 +/- 4.2% vs control 13.57 +/- 3.98%, p < 0.05) and its mRNA expression in endothelial cells (uremic 213.24 +/- 6.13 vs control 200.77 +/- 7.52, p < 0.05). These results suggest that uremic medium alters endothelial function and impairs the antithrombotic functions of cultured endothelial cells. This effect may contribute to the increased cardiovascular and thrombotic risk reported in ESRD patients.

Concentrates containing factor IX could improve haemostasis under conditions of thrombocytopenia: studies in an in vitro model.

BACKGROUND AND OBJECTIVES: We explored the effect on haemostasis of different factor IX (FIX) concentrates under thrombocytopenic conditions using an in vitro perfusion technique. MATERIALS AND METHODS: A moderate experimental thrombocytopenia (25 000-30 000 platelets/microl) was induced by means of a filtration procedure in blood anticoagulated with low-molecular-weight heparin. The effects of three different FIX concentrates - a prothrombin complex concentrate (PCC), an intermediate-purity concentrate (FIX/X), and a high-purity concentrate (HPFIX) - on platelet deposition and fibrin formation on subendothelium were assessed at two different shear rates (600/second and 1200/second). Activation of the coagulation system was monitored through assessment of prothrombin activation fragment 1 + 2 (F1 + 2). RESULTS: Fibrin deposition increased after addition of FIX concentrates, but only showed a significant increase in experiments performed after incubation of PCC at the lower shear rate (600/second) (64.25 +/- 9.61% vs. control 31.22 +/- 8.02%; P < 0.05). Addition of FIX concentrates caused a small increase in the percentage of platelet deposition and area of those aggregates. These differences reached levels of statistical significance in the presence of FIX/X and HPFIX in experiments performed at a shear rate of 600/second. F1 + 2 baseline values in anticoagulated thrombocytopenic blood were 1.15 +/- 0.13 nm and reached levels of 2.49 +/- 0.24 and 3.60 +/- 0.33 nm at shear rates of 600 and 1200/second, respectively. Increments in F1 + 2 observed after addition of different FIX concentrates always remained in the previous ranges. CONCLUSIONS: Data from the present study provide experimental support favouring the concept that FIX concentrates containing other activated factors could improve haemostasis under conditions of moderate thrombocytopenia.

El medio urémico altera las propiedades hemostáticas de células endoteliales humanas en cultivo y aumenta la producción de factor von Willebrand / Uraemic medium alters endothelial function and increases the thrombogenicity and von Willebrand factor expression of the extracellular matrix

El objetivo de este estudio fue determinar la capacidad del suero urémico de modificar las propiedades hemostáticas del endotelio. Para ello se analizó el efecto del medio urémico sobre la morfología y resistencia al flujo de las células endoteliales, así como la trombogenicidad de la matriz subendotelial generada por las células endoteliales.La exposición de células endoteliales en cultivo a un suero urémico indujo alteraciones en su morfología y un crecimiento acelerado de las mismas.Cuando las células endoteliales en cultivo eran expuestas a sangre circulante el desprendimiento de las mismas era superior cuando fueron cultivadas en suero urémico (22 por ciento vs 13 por ciento). La adhesión de plaquetas y la formación de agregados eran superiores en las matrices subendoteliales generadas en presencia de medio urémico (40,23 ñ 6,43 por ciento vs 25,42 ñ 2,69 por ciento, p < 0,05, n = 5).Asimismo, se detectó un aumento en la expresión de antígeno del factor von Willebrand mediante métodos inmunocitoquímicos en las matrices subendoteliales urémicas (17,1 ñ 4,2 por ciento vs 13,57 ñ 3,98, p < 0,05) y la de la expresión de su ARNm en células endoteliales (213,24 ñ 6,13 vs 200,77 ñ 7,52, p < 0,05).Estos resultados indican que el medio urémico altera la función endotelial in vitro y aumenta la trombogenicidad del subendotelio. Estos cambios podrían estar implicados en el aumento del riesgo cardiovascular y de sufrir fenómenos trombóticos que presentan los pacientes con insuficiencia renal crónica. (AU)

Recombinant factor VIIa (Novoseven) restores deficient coagulation: experience from an ex vivo model.

The action of recombinant factor VIIa (rFVIIa) in coagulation deficiencies with increased risk of bleeding was investigated using in vitro perfusion. Blood samples were drawn from healthy donors, a patient with hemophilia A and inhibitors, and six patients undergoing oral anticoagulant treatment. Fragmin 10 U/mL was used as anticoagulant. rFVIIa (10 microg/mL in plasma) was added to blood samples, incubated for 1 minute at 37 degrees C, and perfusion studies performed for 10 minutes at 600 x s(-1) through annular chambers containing damaged vascular segments. Subendothelial fibrin and platelets were expressed as a percentage of subendothelial surface screened. Under different conditions, rFVIIa consistently restored or improved fibrin formation on the damaged vascular subendothelium exposed to circulating blood. It restored fibrin deposition in blood from the hemophilia A patient; in patients undergoing acenocoumarol treatment, it reduced the international normalized ratio (INR) from 2.47 to 1.25 with a significant increase in fibrin deposition. Platelet deposition varied slightly between clinical conditions but was less evident in the hemophilia A patient. These data support the concept that rFVIIa facilitates fibrin formation in these clinical situations, promoting procoagulant activity at sites of vascular damage where tissue factor is exposed. This could improve hemostasis in patients with hemophilia A and inhibitors, and in patients treated with oral anticoagulants.

Uremic medium disturbs the hemostatic balance of cultured human endothelial cells.

We have investigated the ability of serum from uremic patients to modify the thrombogenic properties of the endothelium. The effects of uremic medium on the morphology of endothelial cells (ECs), and their resistance to flow was analyzed. The influence of uremic media on the reactivity of the extracellular matrix (ECM) generated by ECs towards normal platelets was evaluated in a parallel-plate perfusion chamber. Exposure of ECs to uremic medium resulted in abnormal cell morphology and signs of an accelerated growth. Detachment of ECs exposed to circulating blood was increased when cells had been grown with media supplemented with uremic serum (21% vs. 14% non exposed). Platelet deposition was significantly elevated on ECMs generated in the presence of uremic media (uremicECMs) (p<0.01 vs. control studies). Effects of uremic serum were not observed at short incubation periods (5 h) but were evident after 24 or 72 h of incubation. Northern blot analysis revealed increased expression of tissue factor (TF) mRNA in ECs exposed to uremic conditions. Immunocytochemical methods detected an augmented expression of TF antigen on uremic ECMs. Incubation of ECMs with an antibody to human tissue factor prevented the increase in platelet deposition observed in uremic ECMs, suggesting that the presence of TF in ECM could be responsible for the enhanced platelet deposition. Results from our study indicate that uremic medium impairs the antithrombotic functions of cultured endothelial cells.

Prohemorrhagic potential of dipyrone, ibuprofen, ketorolac, and aspirin: mechanisms associated with blood flow and erythrocyte deformability.

Dipyrone, ibuprofen, ketorolac, and aspirin were tested in a well-defined perfusion system (shear rates: 300/s, 800/s, and 1,800/s). Whole blood samples were treated with the drugs at analgesic doses and platelet interaction with damaged subendothelium was measured. All the drugs fully inhibited platelet cyclooxygenase, as assessed by classic aggregometry. Perfusion studies showed that there was a general tendency to reduce the percentage of large aggregates (thrombus; %T), to increase the percentage of adhered platelets (adhesion; %A), and to reduce the height of thrombi with respect to control. Aspirin significantly increased %A and reduced %T at all shear rates tested, whereas dipyrone had the same effect at 800/s, and ketorolac and ibuprofen at 1,800/s. In addition, aspirin significantly reduced erythrocyte deformability with respect to the other drugs. In conclusion, under our experimental conditions, aspirin showed the most remarkable effects on platelet function, closely followed by dipyrone. The effects of ketorolac were moderate, whereas ibuprofen had a minor impact on platelet function.

Antiphospholipid antibodies and thrombophilic factors in giant cell arteritis.

OBJECTIVES: To evaluate the prevalence of thrombophilic risk factors known to induce intravascular clotting and to assess their relationship with ischemic manifestations in giant cell arteritis (GCA). METHODS: Eighty consecutive patients with established GCA were included: 36 with isolated temporal arteritis (TA), 14 with isolated polymyalgia rheumatica (PMR), and 30 with TA and PMR. Forty-four patients (67%) had ischemic phenomena due to GCA. Twelve patients (15%) had thrombotic events unrelated to GCA (6 strokes, 5 deep venous thrombosis, and 1 myocardial infarction). A control group of 100 age- and sex-matched individuals without autoimmune disease, bleeding disorders, thrombosis, or clinical picture of TA or PMR also was analyzed. All participants were tested for the antiphospholipid antibody (aPL) profile, protein C, protein S, antithrombin activity, factor V Leiden mutation, and prothrombin gene G20210A mutation. We also studied fibrinolysis parameters: plasminogen, tissue-type plasminogen activator (t-PA) antigen, t-PA activity, type-1 plasminogen activator inhibitor (PAI-1) antigen, PAI-1 activity, and the 4G/5G polymorphism of the promoter region of the PAI-1 gene. RESULTS: Eleven patients (18%) tested positive for lupus anticoagulant, 24 (30%) for anticardiolipin antibodies, 9 (11%) for anti-beta 2-glycoprotein I antibodies, and 29 (36%) for antiprothrombin antibodies. No relationship was found between these autoantibodies and ischemic manifestations. None of the patients had decreased protein C, protein S or antithrombin activity. Two patients and 2 controls were heterozygous for factor V Leiden, and only 1 patient and 2 controls were heterozygous for the prothrombin gene G20210A mutation. No statistically significant correlation was found between any thrombophilic factor and GCA-related or GCA-unrelated ischemic events. CONCLUSION: GCA patients have a high prevalence of aPL that is not related to ischemic manifestations. Moreover, GCA-related or GCA-unrelated ischemic manifestations do not appear to be due to congenital thrombophilic risk factors. Semin Arthritis Rheum 31:12-20.

Platelet-specific antibodies in HLA-immunized patients receiving chronic platelet support.

BACKGROUND: In HLA-alloimmunized patients, the unexpected failure of HLA-matched platelet transfusions usually raises the suspicion about concomitant platelet-specific antibodies. As the reported frequency of platelet-specific antibodies in multitransfused patients varies widely, the aim of this study was to determine the prevalence of such antibodies in a population of chronic thrombocytopenic patients with HLA antibodies. STUDY DESIGN AND METHODS: From 1985 to 1997, 11,777 determinations of HLA antibodies were performed in 1330 hematologic patients receiving chronic platelet support. Fifty-two patients with HLA alloimmunization that lasted more than 1 month were selected. The search for platelet-specific antibodies was performed by using a monoclonal antibody immobilization of platelet antigens assay, thus allowing the identification of platelet-specific antibodies directed against the platelet glycoproteins (GP) Ib/IX, GPIIb/IIIa, and GPIa/IIa. Specificity of the platelet-specific antibodies was further investigated by using a solid-phase assay with chloroquine-treated platelets. RESULTS: Only 2 (3.8%) of the 52 patients had platelet-specific antibodies. One antibody reacted with an epitope of the GPIIb/IIIa that was present in all the panel platelets, and that probably was an autoantibody. The other was an anti-HPA-5b. CONCLUSIONS: The prevalence of platelet-specific antibodies in patients with HLA alloimmunization is very small. The search for concomitant platelet-specific antibodies would be indicated only when other causes of refractoriness to HLA-matched platelets are ruled out.

Transfusion of incompatible RBCs to a patient with alloanti-Kp(b).

BACKGROUND: The finding of an antibody that reacts against a high-incidence blood group antigen always constitutes a complex transfusion problem because of the difficulty in finding compatible units. When the transfusion of incompatible RBCs is imperative, it would be of great interest to have access to techniques facilitating the prediction of the transfusion outcome. STUDY DESIGN AND METHODS: The case of a patient with alloanti-Kp(b) who required RBC transfusions is reported. The functional activity of this antibody was assessed by both the chemiluminescence test (CLT) and the survival of 51Cr-labeled RBCS: RESULTS: The CLT showed an opsonic index of 0.8 with Kp(b)-positive RBCs (normal values up to 1.6) in pretransfusion studies. During an elective surgical procedure, the patient required the transfusion of one incompatible unit of RBCs, which did not produce hemolysis. Two weeks after this incompatible transfusion, the opsonic index had risen to 11. Results of the 51Cr in vivo study, also performed at that time, indicated 24.3 percent survival of Kp(b)-positive RBCs at 60 minutes and 2.0 percent at 24 hours. CONCLUSION: Results of the CLT correlated with the in vivo transfusion outcome and later with the 51Cr survival study.

Differential response of human and bovine platelets to bovine von Willebrand factor and vascular subendothelium.

Human platelets undergo agglutination when stirred with bovine plasma (BP), but bovine platelets do not. The present study has shown that exposure of washed bovine platelets to subthreshold concentrations of adenosine diphosphate or thrombin before stirring restores their sensitivity to BP, and the cells undergo rapid agglutination. This agglutination was prevented by a monoclonal antibody, to glycoprotein GPIb. Flow cytometry studies revealed that exposure of bovine platelets to thrombin caused an increase in their ability to bind antibodies known to react with human GPIb or GPIIb-IIIa receptors. Interaction of bovine and human platelets with vascular subendothelium revealed additional differences in reactivity. Bovine platelets in citrate anticoagulant reacted poorly with subendothelium under flow conditions compared with human platelets. In contrast, bovine platelets in blood with low molecular weight heparin as anticoagulant adhered more readily than human cells. These findings suggest that different mechanisms are involved in hemostasis in human and bovine species.

Effect of anticoagulants on activation of polymorphonuclear leukocytes induced by shear stress.

We analyzed how actin polymerization, CD11b expression and homotypic aggregation could be used as markers to study leukocyte activation. Leukocytes were obtained from blood anticoagulated with: citrate, unfractioned heparin (UH) and low molecular weight heparin (LMWH). Flow cytometry was used to study actin polymerization and expression of CD11b after leukocyte exposure to shear stress. Leukocyte aggregation was microscopically assessed. Shear increased both actin polymerization and expression of CD11b in citrated blood (100.1±7.1 vs. 85.8±8.5 p< 0.05 and 53.5±3.5 vs. 20.7±5.1; p< 0.005 respectively). These parameters remained unmodified in UH samples. Using both anticoagulants together, we observed increase in CD11b expression induced by shear stress (59.3±2.1 vs. 25.1±11.0; p< 0,05). LMWH samples showed higher basal levels of actin polymerization and CD11b expression than citrated samples (237±40.8, vs. 85.8±8.5 p< 0.05 and 47.8±2.6, vs. 20.7±5.1; p< 0.005) but no changes induced by shear were observed. When LMWH was used in combination with citrate we observed a decrease in basal activation and significant modifications in CD11b expression induced by shear stress (80.0±4.1 vs. 50.4±2.7). Leukocyte aggregation was modified by UH at basal levels and by LMWH after shear stress. These results indicate that exposure to shear stress results in leukocyte activation. The choice of anticoagulant is a crucial factor in studies of leukocyte function.

Platelet procoagulant activity induced in vivo by muromonab-CD3 infusion in uremic patients.

BACKGROUND: Muromonab-CD3 is a murine monoclonal antibody (MoAb) that is used in the prophylaxis and treatment of acute graft rejection. Activation of coagulation and fibrinolysis following anti-CD3 administration have been reported in some patients to lead to irreversible intragraft thrombosis. DESIGN AND METHODS: We have studied the effect of muromonab-CD3 infusion on platelets using flow cytometry in six patients who received three daily doses of muromonab-CD3 as prophylaxis of rejection before receiving a living donor renal transplant. Samples were collected before, 15 and 60 min after muromonab-CD3 infusion. Immunolabeling of platelets was performed in whole blood using dual-color analysis. The following conjugated MoAb were used: anti-CD41a, -CD36, -CD42b, -CD62P, -CD63, -factor V/Va and nonspecific Ig. Samples were analyzed with a FACScan flow cytometer (Becton Dickinson, Mountain View, CA, USA). RESULTS: After muromonab-CD3 infusion, an increase in the binding of MoAb anti-factor V/Va to platelets was seen, which was only statistically significant (2.2% vs. 12.8%, P=.04) after 15 min of the second dose. No significant changes were seen in the other MoAbs studied. No thrombotic complications were observed after transplantation. INTERPRETATION AND CONCLUSION: In uremic patients receiving muromonab-CD3 infusion as prophylaxis of graft rejection, an increase in the binding of anti-factor V/Va, denoting an increased exposure of anionic phospholipids in platelets, was seen. This increase in platelet procoagulant activity might contribute to the appearance of thromboses within renal graft seen in some patients who received muromonab-CD3.

Comparison of the effects of human erythrocyte ghosts and intact erythrocytes on platelet interactions with subendothelium in flowing blood.

We investigated whether ghosts behaved similarly to intact erythrocytes to maintain regular primary hemostasis under flow conditions. To this end we performed perfusion experiments with whole blood in which erythrocytes were replaced by pink ghosts, and platelet interaction with the subendothelial surface of a damaged vessel was morphometrically evaluated. The same objective was sought by means of studies with a platelet function analyzer (PFA-100(TM) instrument). Perfusions performed with control blood reconstituted with intact erythrocytes gave rise to 0.4+/-0.2% contact but not spread platelets, 10.8+/-3.4% adhering and spread platelets, 16.3+/-4.6% platelets in thrombi, with 27.5+/-7.4% of the surface covered. Even though the average diameter of the ghosts was smaller than that of intact erythrocytes (5.3 microm vs. 7.7 microm), the values obtained in perfusions performed with ghosts were similar to those of the erythrocyte controls. Studies performed with the PFA-100(TM) analyzer were consistent with those observed in perfusion studies. The viscosity of control blood was compared with that of blood reconstituted with ghosts. At shear rates lower than 450 s(-1), the viscosity of the ghost samples was higher than that of the controls, but the difference progressively decreased as shear rate increased up to 750 s(-1) (3.61+/-0.15 and 3.71+/-0.17 cP, respectively). In conclusion, the results of our study showed that ghosts behaved similarly to intact erythrocytes in maintaining a normal platelet interaction with digested subendothelium, under conditions of moderate shear rate and constant hematocrit (40%). The rheological activity of ghosts, bodies that are metabolically less active, was sufficient for them to satisfactorily act as substitutes for intact erythrocytes in our system.