Effects of nitrite and far-red light on coagulation.

Nitric oxide, NO, has been explored as a therapeutic agent to treat thrombosis. In particular, NO has potential in treating mechanical device-associated thrombosis due to its ability to reduce platelet activation and due to the central role of platelet activation and adhesion in device thrombosis. Nitrite is a unique NO donor that reduces platelet activation in that it's activity requires the presence of red blood cells whereas NO activity of other NO donors is blunted by red blood cells. Interestingly, we have previously shown that red blood cell mediated inhibition of platelet activation by adenosine diphosophate (ADP) is dramatically enhanced by illumination with far-red light that is likely due to photolysis of red cell surface bound NO congeners. We now report the effects of nitrite, far-red light, and their combination on several measure of blood coagulation using a variety of agonists. We employed turbidity assays in platelet rich plasma, platelet activation using flow cytometry analysis of a fluorescently labeled antibody to the activated platelet fibrinogen binding site, multiplate impedance-based platelet aggregometry, and assessment of platelet adhesion to collagen coated flow-through microslides. In all cases, the combination of far-red light and nitrite treatment decreased measures of coagulation, but in some cases mono-treatment with nitrite or light alone had no effect. Perhaps most relevant to device thrombosis, we observed that platelet adhesions was inhibited by the combination of nitrite and light treatment while nitrite alone and far-red light alone trended to decrease adhesion, but the results were mixed. These results support the potential of combined far-red light and nitrite treatment for preventing thrombosis in extra-corporeal or shallow-tissue depth devices where the far-red light can penetrate. Such a combined treatment could be advantageous due to the localized treatment afforded by far-red light illumination with minimal systemic effects. Given the role of thrombosis in COVID 19, application to treatment of patients infected with SARS Cov-2 might also be considered.

Erythrocytic bioactivation of nitrite and its potentiation by far-red light.

BACKGROUND: Nitrite is reduced by heme-proteins and molybdenum-containing enzymes to form the important signaling molecule nitric oxide (NO), mediating NO signaling. Substantial evidence suggests that deoxygenated hemoglobin within red blood cells (RBCs) is the main erythrocytic protein responsible for mediating nitrite-dependent NO signaling. In other work, infrared and far red light have been shown to have therapeutic potential that some attribute to production of NO. Here we explore whether a combination of nitrite and far red light treatment has an additive effect in NO-dependent processes, and whether this effect is mediated by RBCs. METHODS AND RESULTS: Using photoacoustic imaging in a rat model as a function of varying inspired oxygen, we found that far red light (660 nm, five min. exposure) and nitrite feeding (three weeks in drinking water at 100 mg/L) each separately increased tissue oxygenation and vessel diameter, and the combined treatment was additive. We also employed inhibition of human platelet activation measured by flow cytometry to assess RBC-dependent nitrite bioactivation and found that far red light dramatically potentiates platelet inhibition by nitrite. Blocking RBC-surface thiols abrogated these effects of nitrite and far-red light. RBC-dependent production of NO was also shown to be enhanced by far red light using a chemiluminescence-based nitric oxide analyzer. In addition, RBC-dependent bioactivation of nitrite led to prolonged lag times for clotting in platelet poor plasma that was enhanced by exposure to far red light. CONCLUSIONS: Our results suggest that nitrite leads to the formation of a photolabile RBC surface thiol-bound species such as an S-nitrosothiol or heme-nitrosyl (NO-bound heme) for which far red light enhances NO signaling. These findings expand our understanding of RBC-mediated NO production from nitrite. This pathway of NO production may have therapeutic potential in several applications including thrombosis, and, thus, warrants further study.

High-frequency (13.56-MHz) and ultrahigh-frequency (915-MHz) radio identification systems do not affect platelet activation and functions.

BACKGROUND: In radiofrequency identification (RFID) systems used in labeling of blood components, blood cells are subjected to the direct influence of electromagnetic waves throughout the storage period. The aim of this study was to prove the safety of storage of platelet concentrates (PCs) in containers labeled with RFID tags. STUDY DESIGN AND METHODS: Ten pooled PCs obtained from 12 buffy coats each suspended in additive solution were divided into three separate containers that were assigned to three groups: control, PCs labeled with ultrahigh frequency (UHF) range tags and exposed to 915-MHz radio waves, and PCs labeled with high-frequency (HF) range tags and exposed to 13.56-MHz radio waves. PCs were stored at 20 to 24°C for 7 days. In vitro tests of platelet (PLT) function were performed on the first, fifth, and seventh days of storage. RESULTS: There were no significant differences in pH; hypotonic shock resistance; surface expression of CD62P, CD42a, or CD63; release of PLT-derived microparticles; PLT aggregation; and number of PLTs between PCs stored at a constant exposure to radio waves of two different frequencies and the control group on the first, fifth, and seventh days of storage. CONCLUSION: The results of the study indicate no impact of electromagnetic radiation generated in HF and UHF RFID systems and constant contact with the tags on the quality of stored PCs.

Imparting electroactivity to polycaprolactone fibers with heparin-doped polypyrrole: Modulation of hemocompatibility and inflammatory responses.

Hemocompatibility, anti-inflammation and anti-thrombogenicity of acellular synthetic vascular grafts remains a challenge in biomaterials design. Using electrospun polycaprolactone (PCL) fibers as a template, a coating of polypyrrole (PPy) was successfully polymerized onto the fiber surface. The fibers coated with heparin-doped PPy (PPy-HEP) demonstrated better electroactivity, lower surface resistivity (9-10-fold) and better anti-coagulation response (non-observable plasma recalcification after 30min vs. recalcification at 8-9min) as compared to fibers coated with pristine PPy. Red blood cell compatibility, measured by% hemolysis, was greatly improved on PPy-HEP-coated PCL in comparison to uncoated PCL (3.9±2.1% vs. 22.1±4.1%). PPy-HEP-coated PCL fibers also exhibited higher stiffness values (6.8±0.9MPa vs. 4.2±0.8MPa) as compared to PCL fibers, but similar tensile strengths. It was also observed that the application of a low alternating current led to a 4-fold reduction of platelet activation (as quantitated by CD62p expression) for the PPy-HEP-coated fibers as compared to non-stimulated conditions. In parallel, a reduction in the leukocyte adhesion to both pristine PPy-coated and PPy-HEP-coated fibers was observable with AC stimulation. Overall, a new strategy involving the use of hemocompatible conducting polymers and electrical stimulation to control thrombogenicity and inflammatory responses for synthetic vascular graft designs was demonstrated.

Evaluation of the effects of preconditioning regimens on hepatic veno-occlusive disease in mice after hematopoietic stem cell transplantation.

Pre-conditioning regimens before hematopoietic stem cell transplantation (HSCT), such as total body irradiation (TBI) or busulfan/cyclophosphamide (BU/CY), are associated with hepatic veno-occlusive disease (HVOD). However, the mechanism of these regimens on hepatic veno-occlusive disease remains unclear. The aim of this study is to evaluate the effect of TBI or BU/CY on HVOD in mice after HSCT. Mice received TBI or BU/CY followed by HSCT. Analysis of liver pathology and function, and platelet aggregation were performed. Both these regimens caused damage to liver sinusoid endothelial cells, leading to loss of normal structural integrity of liver sinusoid, abnormal liver function, fibrin deposition, inflammatory cells infiltration and platelet aggregation. No differences of liver function in these regimens were observed. Increased hepatic lipid droplets, mitochondrial swelling and higher incidence of HVOD were observed in BU/CY. In conclusion, both TBI and BU/CY caused damage to liver sinusoid endothelial cells and occurrence of HVOD with higher incidence for BU/CY. Meanwhile, inflammation and platelet activation was also observed, suggesting targeting them maybe beneficial in the prophylaxis of HVOD.

Induction of monocyte-to-dendritic cell maturation by extracorporeal photochemotherapy: initiation via direct platelet signaling.

Extracorporeal Photochemotherapy (ECP) is a widely used therapy for cutaneous T cell lymphoma (CTCL). Although the mechanism of clinical action of ECP is not precisely established, previous studies have shown evidence of induction of dendritic cells (DCs). Here we show that, under flow conditions similar to those in post-capillary venules, ECP promotes platelet immobilization and activation, initiating stepwise receptor-ligand interactions with monocytes, which then differentiate into DC. These findings clarify how ECP directly stimulates DC maturation; suggest a new clinically applicable approach to the obtainment of DC; and identify a novel mechanism that may reflect physiological induction of DC.

Analysis of reasons for not implementing pathogen inactivation for platelet concentrates.

In the last 10 years three technologies capable of inactivating pathogens in platelet concentrates have been authorized in Europe although only one based on the addition of amotosalen and illumination with ultraviolet A (UVA) light, has been approved by the National Agency for the Safety of Medicines and Health Products (ANSM). An intense debate exists about the implementation of pathogen inactivation technologies for labile blood components in general and for platelet concentrates in particular. In this review, we will analyze some of the most frequently argued reasons for not implementing pathogen inactivation for platelet components, i.e.: current platelet components are safe enough; pathogen inactivation technologies might be toxic for the recipient; and pathogen inactivation technologies affect platelet function and increase the risk of bleeding. The analysis and discussion of the evidence currently available to answer those reservations will be limited to the pathogen inactivation technology based on amotosalen and UVA.

Primary hemostatic capacity of whole blood: a comprehensive analysis of pathogen reduction and refrigeration effects over time.

BACKGROUND: Whole blood (WB) has been used in combat since World War I as it is readily available and replaces every element of shed blood. Component therapy has become standard; however, recent military successes with WB resuscitation have revived the debate regarding wider WB use. Characterization of optimal WB storage is needed. We hypothesized that refrigeration preserves WB function and that a pathogen reduction technology (PRT) based on riboflavin and ultraviolet light has no deleterious effect over 21 days of storage. STUDY DESIGN AND METHODS: WB units were stored for 21 days either at 4°C or 22°C. Half of each temperature group underwent PRT, yielding four final treatment groups (n = 8 each): CON 4 (WB at 4°C); CON 22 (WB at 22°C); PRT 4 (PRT WB at 4°C); and PRT 22 (PRT WB at 22°C). Testing was at baseline, Days 1-7, 10, 14, and 21. Assays included coagulation factors; platelet activation, aggregation, and adhesion; and thromboelastography (TEG). RESULTS: Prothrombin time (PT) and partial thromboplastin time increased over time; refrigeration attenuated the effects on PT (p ≤ 0.009). Aggregation decreased over time (p ≤ 0.001); losses were attenuated by refrigeration (p ≤ 0.001). Refrigeration preserved TEG parameters (p ≤ 0.001) and PRT 4 samples remained within normal limits throughout the study. Refrigeration in combination with PRT inhibited fibrinolysis (p ≤ 0.001) and microparticle formation (p ≤ 0.031). Cold storage increased shear-induced platelet aggregation and ristocetin-induced platelet agglutination (p ≥ 0.032), as well as GPIb-expressing platelets (p ≤ 0.009). CONCLUSION: The in vitro hemostatic function of WB is largely unaffected by PRT treatment and better preserved by cold storage over 21 days. Refrigerated PRT WB may be suitable for trauma resuscitation. Clinical studies are warranted.

Pathogen inactivation of platelets using ultraviolet C light: effect on in vitro function and recovery and survival of platelets.

BACKGROUND: We evaluated the effect of treating platelets (PLTs) using ultraviolet (UV)C light without the addition of any photosensitizing chemicals on PLT function in vitro and PLT recovery and survival in an autologous radiolabeled volunteer study. STUDY DESIGN AND METHODS: For in vitro studies, pooled or single buffy coat-derived PLT concentrates (PCs) were pooled and split to obtain identical PCs that were either treated with UVC or untreated (n = 6 each) and stored for 7 days. PLT recovery and survival were determined in a two-arm parallel autologous study in healthy volunteers performed according to BEST guidelines. UVC-treated or untreated PCs (n = 6 each) were stored for 5 days and were compared to fresh PLTs from the same donor. RESULTS: There were no significant differences on Day 7 of storage between paired UVC-treated and control PC units for pH, adenosine triphosphate, lactate dehydrogenase, CD62P, CD63, PLT microparticles, and JC-1 binding, but annexin V binding, lactate accumulation, and expression of CD41/61 were significantly higher in treated units (p < 0.05). Compared with control units, the recovery and survival of UVC-treated PC were reduced after 5 days of storage (p < 0.05) and when expressed as a percentage of fresh values, survival was reduced by 20% (p = 0.005) and recovery by 17% (p = 0.088). CONCLUSION: UVC-treated PLTs stored for 5 days showed marginal changes in PLT metabolism and activation in vitro and were associated with a degree of reduction in recovery and survival similar to other pathogen inactivation systems that are licensed and in use.

Does bipolar pacemaker current activate blood platelets?

OBJECTIVE: The aim of this study was to investigate whether bipolar pacemaker current lead can activate blood platelets. The null hypothesis was that 1 minute of electrical stimulation of platelets would not influence their subsequent reactivity to adenosine diphosphate (ADP). BACKGROUND: Both platelets and muscle cells contain actin and myosin filaments, and both cells are activated following calcium influx. Muscle cells open their calcium channels and contract when exposed to an electric current. Current through a bipolar pacemaker lead will expose a small volume of blood, including platelets, to the depolarizing current. Platelet activation may ensue, resulting in aggregation, release reaction, and contraction. In contrast, a unipolar pacemaker system will not depolarize blood, but transmit current directly into the myocardium, and the current afterward passes through other tissues before returning to the pacemaker can. METHODS: Platelet-rich plasma was prepared from two healthy subjects. Platelet reactivity to the agonist ADP was tested in paired samples in an aggregometer in a case/control setup. RESULTS: Eighteen of 46 tested pairs of platelet-rich plasma showed increased reactivity in the paced sample; 26 were unchanged while two showed decreased reactivity in the paced sample. Using a two-sided sign test, the null hypothesis was rejected (P = 0.0004). CONCLUSIONS: The study demonstrates increased reactivity to ADP in platelets exposed in vitro to stimulation by pacemaker current. The clinical relevance of these findings remains to be investigated.

In vitro evaluation of metabolic changes and residual platelet responsiveness in photochemical treated and gamma-irradiated single-donor platelet concentrates during long-term storage.

BACKGROUND: Photochemical treatment (PCT) prevents replication of pathogens in platelet concentrates (PCs) by cross-linking nucleic acids and thus affects all cells containing DNA or RNA. STUDY DESIGN AND METHODS: Fourteen double-dose single-donor PCs were divided into two study arms. The double-dose PCs were split in two identical units, PCT and conventional control PCs. Study Arm A consisted of seven PCT PCs with corresponding untreated controls, whereas Study Arm B consisted of seven PCT PCs with corresponding gamma-irradiated control. Metabolic changes and agonist-induced platelet (PLT) response were evaluated during storage for up to 12 days. RESULTS: Higher rate of PLT destruction, illustrated by reduced PLT content, increased lactate dehydrogenase levels, and higher CD61+ microparticle formation rate, were observed after PCT. Generally PCT accelerated metabolic changes in PCs and reduced agonist-induced (collagen or thrombin receptor activator peptide [TRAP]) aggregation responses. Flow cytometric analysis of CD62P and CD42b (GPIbalpha) expression showed higher spontaneous PLT activation in PCT PCs from 5 days of storage. Correspondingly, a reduced capacity for up regulation of CD62P expression and down regulation of CD42b was observed in PCT PLTs after stimulation by the agonists ADP or TRAP. CONCLUSION: Generally reduced in vitro PLT quality was observed after PCT during storage for up to 12 days, with marked reduction from 5 days of storage. Compared to conventional PCs, reduced agonist-induced aggregation and glycoprotein expression were observed after PCT during storage, corresponding to significantly higher level of spontaneous PLT activation in PCT PCs. Clinical studies of efficacy and safety of PCT PCs stored for more than 5 days are recommended.

Platelet function after single [153Sm]EDTMP therapy in prostate cancer.

AIM: Aim of the study was to assess whether [153Sm] EDTMP therapy at a low-dose is associated with platelet activation. METHODS: In 29 patients suffering from metastatic prostate cancer platelet count and various platelet function parameters have been monitored for 2 months after a single (the first) application of 1.1 GBq mCi [153Sm]EDTMP. RESULTS: After 3 days insignificant signs of platelet activation (increase in malondialdehyde, adenosine diphosphate-induced platelet aggregation, decreased platelet sensitivity) occur, normalizing rapidly. At the nadir of platelet count (3-4 weeks) platelet aggregation response in non-count adjusted samples is somewhat lower, while activity per cell (count adjusted samples) is unchanged. Platelet proteins do not change at all. Insignificant activation of platelet function at day 3 is interpreted as an indicator of mild oxidation injury, late aggregation response changes in non-adjusted samples seem only to reflect temporarily decreased number of circulating platelets. Samarium-153-EDTMP therapy at doses (1.1 GBq) used in the Vienna-protocol is not associated with a significantly altered functional behavior of platelets. CONCLUSIONS: We conclude that a single dose of 1.1 GBq 153Sm-EDTMP does not significantly affect in vivo and ex vivo platelet function.

Regeneration of megakaryocytopoiesis and thrombopoiesis in vitro from X-irradiated human hematopoietic stem cells.

In the present study, we investigated whether X-irradiated hematopoietic stem cells can be induced to undergo megakaryocytopoiesis and thrombopoiesis in vitro using cytokine combinations that have been demonstrated to be effective for conferring increased survival on irradiated human CD34(+) megakaryocytic progenitor cells (colony-forming unit megakaryocytes; CFU-Meg), such as thrombopoietin (TPO), interleukin 3 (IL3), stem cell factor and FLT3 ligand. Culture of nonirradiated CD34(+) cells in serum-free medium supplemented with multiple cytokine combinations led to an approximately 200- to 600-fold increase in the total cell numbers by day 14 of culture. In contrast, the growth of X-irradiated cells was observed to be one-sixth to one-tenth that of the nonirradiated cultures. Similarly, total megakaryocytes were increased by 50- to 130-fold, while culture of X-irradiated cells yielded one-fourth to one-eighth of the control numbers. At this time, CD41(+) particles, which appeared to be platelets, were produced in the medium harvested from nonirradiated and irradiated cultures. Although radiation suppressed cell growth and megakaryocytopoiesis, there were no significant differences in thrombopoiesis between the two types of culture. These results suggest that X-irradiated CD34(+) cells can be induced to undergo nearly normal terminal maturation through megakaryocytopoiesis and thrombopoiesis by stimulation with appropriate cytokine combinations.

Evaluation of platelet mitochondria integrity after treatment with Mirasol pathogen reduction technology.

BACKGROUND: Previous studies showed that Mirasol (Navigant Biotechnologies, Inc.) pathogen reduction technology (PRT) treatment resulted in an increase in platelet (PLT) glucose consumption and lactate production rates and decrease in pH in media during PLT storage. Increased glycolytic flux could result from damage to mitochondria and/or increased ATP consumption. STUDY DESIGN AND METHODS: PLT concentrates were collected by standard automated blood component collection system (Trima, Gambro BCT) procedure on Day 0 and treated with Mirasol PRT treatment on Day 1. PLT mitochondrial transmembrane potential was evaluated by staining PLTs with JC-1 followed by flow cytometry analysis. Mitochondrial enzymatic activity was measured by the MTT assay. ATP content and pH were also quantified. The values for these measurements were compared among control, untreated, and pathogen reduction technology (PRT)-treated PLTs during PLT storage for up to 7 days. RESULTS: No significant changes were found in pH, JC-1 signal, MTT activity, and ATP content of the PLTs immediately after PRT treatment. The treated PLTs exhibited a moderate but significantly accelerated decrease in pH and lower ATP content after 7-day storage when compared to control PLTs. Neither the JC-1 assay nor the MTT assay, however, showed a significant difference between control and treated PLTs during PLT storage. CONCLUSIONS: There is no evidence from these studies that Mirasol PRT treatment alters PLT mitochondrial structural and functional integrity immediately after treatment and during PLT storage. An increased demand for ATP may be the driving force for observed increases in both the glycolytic flux and the oxidative metabolism observed in treated PLTs.

[Effect of SWF-radiation on thrombocytes and erythrocyte functions of albino rats upon stress condition].

With the help of a specially designed generator, we have investigated the effect of electromagnetic SWF-oscillation, at nitric oxide molecular spectrum of radiation and absorption, on the function of thrombocytes and erythrocytes of albino rats in the state of immobilizing stress. 5, 15 and 30 min long SWF-radiation treatments were demonstrated to foster various degrees of restoration of thrombocyte and erythrocyte function, the efficiency depending on the period of radiation. It was after a 30 min radiation of rats that a most expressed restoration of thrombocyte and erythrocyte functional activity was observed.

Catheter based intracoronary brachytherapy leads to increased platelet activation.

BACKGROUND: Vascular brachytherapy (VBT) after percutaneous coronary intervention (PCI) is associated with a higher risk of stent thrombosis than conventional treatment. OBJECTIVE: To investigate in vivo periprocedural platelet activation with and without VBT, and to assess a possible direct effect of radiation on platelet activation. DESIGN: Of 50 patients with stable angina, 23 received VBT after PCI, while 27 had PCI only. The 23 patients who received VBT after PCI were pretreated for one month with aspirin and clopidogrel. Platelet activation was assessed by flow cytometry. RESULTS: The two patient groups did not differ in their platelet activation before the intervention. There was a significant increase in activation immediately after VBT, with 21.2% (interquartile range 13.0% to 37.6%) thrombospondin positive and 54.0% (42.3% to 63.6%) CD 63 positive platelets compared with 12.7% (9.8% to 14.9%) thrombospondin positive and 37.9% (33.2% to 45.2%) CD 63 positive platelets before the intervention (p < 0.001 and p < 0.01, respectively). Patients without VBT had no periprocedural difference in platelet activation immediately after PCI. No increase in platelet activation was found after ex vivo irradiation of blood samples obtained from healthy controls. CONCLUSIONS: Catheter based intracoronary VBT carried out according to current standards is highly thrombogenic. The current antithrombotic treatment with aspirin and clopidogrel is not sufficient to suppress platelet activation during the procedure. From in vitro experiments, it appears that platelet activation during brachytherapy is not caused by irradiation but by the procedure of catheter based VBT.

Irradiation increases the interactions of platelets with the endothelium in vivo: analysis by intravital microscopy.

Adhesion of platelets to the endothelium is believed to be a major factor contributing to thrombosis and vascular occlusion after radiotherapy or endovascular irradiation. In the present study, platelet-endothelium interactions were analyzed in vivo by intravital microscopy in mesenteric venules of mice according to three parameters: (1) platelet rolling, (2) platelet adhesion, and (3) the presence of platelet clusters. A 10-Gy total-body irradiation of mice resulted in an increase in the frequency of appearance of these three types of platelet-endothelium interactions in postcapillary venules 6 and 24 h after exposure, whereas only minor alterations were seen in large venules. In addition, the duration of platelet adhesion was increased 24 h after irradiation in both postcapillary and large venules. However, P-selectin was not up-regulated on the platelet membrane and platelet-leukocytes were not seen rolling together, suggesting that changes in platelet-endothelial cell interaction result from endothelial cell activation rather than platelet activation. Our data suggest that irradiation transforms resting endothelial cells to a pro-adhesive surface for platelets, which could ultimately lead to thrombosis.

Effects of X-ray radiation on the rheologic properties of platelets and lymphocytes.

BACKGROUND: Blood is often irradiated before transfusion for severely immunocompromised patients to prevent a potentially fatal complication of transfusion-associated GVHD. STUDY DESIGN AND METHODS: This study evaluates the effects of X-ray radiation on platelet and lymphocyte rheology because the ability of these blood cells to deform is vital to their flow throughout the microvascular system. Micropipette aspiration experiments were conducted on platelets and lymphocytes exposed to X-ray radiation doses of 0 (control), 25, and 50 Gy. RESULTS: A significant increase in the Young modulus of elasticity was observed between control platelets and irradiated platelets at 25 Gy (p = 0.02) and 50 Gy (p = 0.03). Percent cell activation increased significantly in 25 Gy-irradiated platelets (p = 0.008). In addition, lymphocytes irradiated at 25 Gy have a higher viscosity than controls (p < 0.02). A significantly larger number of activated cells is found in the 50 Gy-irradiated lymphocyte population (p < 0.04). CONCLUSION: The changes in the deformability and activation of irradiated platelets and lymphocytes may reduce local blood flow and lead to intermittent blockage, which may cause a change of blood flow in microvasculatures.

Ticlopidine inhibits the prothrombotic effects of thrombopoietin and ameliorates survival after supralethal total body irradiation.

Thrombopoietin modulates the response of platelets to several agonists and, on the other hand, those agonists can be released following irradiation. Thus, we have determined the effects of thrombopoietin, on its own and in combination with ticlopidine, an anti-platelet drug, on platelet activation, thrombosis formation and survival of irradiated C57BL6/J mice. Administration of thrombopoietin 2 h after 9 Gy total body irradiation increased the 125I-fibrin deposition in mouse tissues and accelerated platelet consumption as revealed by an enhanced drop in platelet counts. Additionally, the number of activated platelets, i.e. those expressing P-selectin on their membrane, was higher in thrombopoietin-treated mice as compared to the placebo group, regardless ex vivo stimulation with agonists. These effects of thrombopoietin on platelet activation and consumption were reduced when mice were pretreated with ticlopidine. The combination of ticlopidine with thrombopoietin almost fully promoted 180-day survival, reaching the same efficacy as bone marrow transplantation, while only 30% of the mice treated with thrombopoietin alone survived. In summary, thrombopoietin induces long term-mortality of irradiated mice probably through platelet-mediated thrombosis and thus, ticlopidine efficiently counteracts these adverse effects of thrombopoietin.