Lactadherin as a Probe for Phosphatidylserine Exposure and as An Anticoagulant for the Procoagulant Activity in the Study of Stored Platelets.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3150-3150
Author(s):  
Jin Zhou ◽  
Jinxiao Hou ◽  
Wen Li ◽  
Xiaoqian Zhang ◽  
Yueyue Fu ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Confocal Microscopy ◽  
Conflicts Of Interest ◽  
Annexin V ◽  
Progressive Increase ◽  
Procoagulant Activity ◽  
Platelet Concentrates ◽  
Time Analysis ◽  
Coagulation Time ◽  
Enzymatic Assays

Abstract Abstract 3150 Poster Board III-87 Background Phosphatidylserine (PS) can support coagulant reactions. However, it is uncertain how the location and extent of PS exposure to the membranes of stored platelets affect such reactions. We compared annexin V with lactadherin as a way of detecting how of PS exposure influences the procoagulant properties of stored platelets in platelet concentrates (PCs). Method PS exposure and the relevant procoagulant activity (PCA) of platelets in 5 different PCs were investigated by flow cytometry, confocal microscopy, coagulation time analysis and enzymatic assays. PS exposure was separately measured using annexin V and lactadherin, respectively. Results Exposure of PS to stored platelets promoted thrombin formation. A progressive increase in PS exposure was detected by flow cytometry. Moreover, using lactadherin, we identifed higher levels of PS exposure on the platelets and platelet-derived microparticles (PMPs) compared to detection using annexin V. The percentage of PS-positive cells was 0.02 % by annexin V versus 0.3 % by lactadherin on day 0, 7.5 % by annexin V versus 12.3 % by lactadherin on day 5, and 29 % by annexin V versus 44.3 % by lactadherin on day 9. Rare microparticles (MPs) were released from fresh platelets, and, the number of PMPs increased approximately 2-fold on day 5 and further progressively increased. Using lactadherin and platelets in the earlier stage of storage, confocal microscopy revealed earlier and localized PS exposure based on plasma membrane staining. For later storage platelets, increased levels of PS-positive platelets and PMPs were clearly detected by both annexin V and lactadherin. Thirty-two nM lactadherin or annexin V prolonged coagulation time 2.4 fold versus 2 fold. The productions of thrombin and intrinsic/extrinsic factor Xase were approximately inhibited 85 % and 60 % by lactadherin and annexin V, respectively. Conclusion PS exposure was localized to the cellular rims, blebbing vesicles and thin elongated filopodia-like areas on banked platelets. Furthermore, lactadherin provides a more accurate measurement of PS exposure and the relevant with PCA, which is an important factor to consider for transfusion medicine. Our findings of elevated PS-positive platelets and PMPs indicate that platelets should not be stored for extended periods of time. Disclosures No relevant conflicts of interest to declare.

scholarly journals Procoagulant Activity of Blood and Endothelial Cells via Phosphatidylserine Exposure and Microparticle Delivery in Patients with Diabetic Retinopathy

2018 ◽  
Vol 45 (6) ◽  
pp. 2411-2420 ◽  
Author(s):  
Ying Su ◽  
Jingli Chen ◽  
Zengxiang Dong ◽  
Yan Zhang ◽  
Ruishuang Ma ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Endothelial Cells ◽  
Diabetic Retinopathy ◽  
Confocal Microscopy ◽  
Blood Cells ◽  
Procoagulant Activity ◽  
Diabetic Patients ◽  
Healthy Controls ◽  
Clotting Time ◽  
Coagulation Time

Background/Aims: The mechanisms for thrombosis in diabetic retinopathy (DR) are complex and need to be further elucidated. The purpose of this study was to test phosphatidylserine (PS) exposure on microparticles (MPs) and MP-origin cells from the circulation and to analyze cell-/MP-associated procoagulant activity (PCA) in DR patients. Methods: PS-positive MPs and cells from healthy controls (n = 20) and diabetic patients (n = 60) were analyzed by flow cytometry and confocal microscopy. Clotting time and purified coagulation complex assays were used to measure PCA. Results: PS exposure on platelets and monocytes was higher in proliferative DR (PDR) patients than in non-PDR patients or controls. The highest levels of MPs (derived from platelets [30%], erythrocytes [13%], leukocytes [28%], and endothelial cells [10%]) were found in patients with PDR. In addition, PS exposure on blood cells and shed MPs in DR patients led to significantly increased FXa and FIIa generation, fibrin formation, and markedly shortened coagulation time. Moreover, lactadherin reduced 70% of PCA by blocking PS, while an anti-tissue factor antibody had a smaller effect. Conclusion: Our results confirmed that PCA in DR patients may be partly ascribed to PS exposure and MP release from blood and endothelial cells. Lactadherin may act as an efficient anticoagulant factor in this process.

Preclinical Development Of a Nanomedicne Approach For Multiple Myeloma Targeting The Myc Oncoprotein

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4228-4228
Author(s):  
Deepti Soodgupta ◽  
Dipanjan Pan ◽  
Grace Hu ◽  
Angana Senpan ◽  
Xiaoxia Yang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Drug Delivery ◽  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Conflicts Of Interest ◽  
Annexin V ◽  
Free Drug ◽  
Preclinical Development ◽  
Late Antigen ◽  
Mtt Assays

Abstract Purpose This study investigated alpha 4 beta 1/ Very Late Antigen-4 (α4β1/ VLA-4)-integrin targeted nanotherapy approach to deliver a new lipase-labile prodrug. Experimental Design A phospholipid-based MYC-MAX inhibitor prodrug (MI1-PD) was synthesized, and its inherent anti-proliferate potency was compared to the lipid-free compound (MI1) using mouse multiple myeloma (MM) cell line (5TGM1). VLA-4-targeted perfluorocarbon (PFC) nanoparticles binding to 5TGM1 cells was measured and compared to biomarker expression assessed with flow cytometry using antibodies. The efficacy of MI1-PD incorporated into non-targeted and VLA-4-targeted PFC NP exposed to 5TGM1 cells was assessed with MTT assays, Annexin V and cell cycle analysis. Results MI1-PD was more potent by several orders of magnitude than its free drug counterpart in culture. Targeted NP binding correlated well with biomarker expression assessment by flow cytometry in 5TGM1 cells. Non-targeted NPs had no appreciable binding to 5TGM1 cells. High anti-MM potency of MI1-PD was noted in VLA-4-targeted NPs compared to the non-targeted NPs demonstrating that the efficacy was dependent on expression of the targeted biomarker to afford particle-to-cell drug delivery. Conclusions These results suggest the feasibility of an improved integrin VLA-4- targeted nanotherapy approach to deliver a lipase- labile prodrug construct, MI1-PD. Disclosures: No relevant conflicts of interest to declare.

Combined Inhibition of Wee1 and Poly(ADP Ribose) Polymerase1/2 Synergistically Inhibits Acute Myeloid Leukemia Cells By Enhancing DNA Damage and the Induction of Apoptosis

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3621-3621 ◽  
Author(s):  
Jonathan C Snedeker ◽  
Tamara M Burleson ◽  
Raoul Tibes ◽  
Christopher C. Porter
Keyword(s):  
Flow Cytometry ◽  
Cell Proliferation ◽  
Dna Damage ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Combination Index ◽  
Annexin V ◽  
Concomitant Treatment ◽  
Western Blots

Abstract Introduction: Successful treatment of AML remains dependent upon cytotoxic chemotherapy. However, traditional regimens are not well tolerated by older patients who are at highest risk of disease, and salvage rates after relapse are low, necessitating novel therapeutic strategies. Our groups identified Wee1 as a potential therapeutic target in AML, particularly in the context of concomitant treatment with cytarabine (Tibes et al, Blood, 2012; Porter et al, Leukemia, 2012). Wee1 inhibits CDK1&2 via phosphorylation thereby stalling cell cycle progression. One consequence of Wee1 inhibition/CDK1 activation is impairment of DNA repair via homologous recombination (Krajewska et al, Oncogene, 2013). Cells in which HR is impaired are dependent upon Parp1/2 function, and HR deficient cells are particularly sensitive to Parp1/2 inhibition. Therefore, we hypothesized that combined Wee1 and Parp1/2 inhibition may result in greater inhibition of AML cell proliferation and survival than either alone. Methods: Human AML cell lines, MV4-11 and Molm-13, and a mouse AML that expresses MLL-ENL/FLT3-ITD were cultured with various concentrations of a Wee1 inhibitor (AZ1775) and a Parp1/2 inhibitor (olaparib) and counted 72 hours later by propidium iodide exclusion and flow cytometry. In some experiments, cells were split into fresh media to recover for 72 more hours. Combination Index (CI) values were calculated by the method of Chou and Talalay. Apoptosis was measured using Annexin V/7AAD and flow cytometry. Western blots were used to confirm inhibition of CDK1/2 phosphorylation and to measure DNA damage induction (gamma-H2AX). Results: Combined inhibition of Wee1 and Parp1/2 was synergistic, as measured by cell numbers at 72 hours, in all 3 cell lines tested, with combination index values ranging from 0.3 to 0.9. When cells were allowed to recover after treatment, those treated by single agents were able to continue proliferating. However, those treated with the combination did not recover as well or at all, indicating greatly impaired proliferative capacity. Combined inhibition of Wee1 and Parp1/2 also resulted in a significant increase in apoptosis greater than either drug alone. Western blots for gamma-H2AX confirmed that the combination of Wee1 and Parp1/2 resulted in more DNA damage than either drug alone. Discussion: Combined inhibition of Wee1 and Parp1/2 results in greater inhibition of AML cell proliferation, DNA damage and apoptosis than either drug alone. Future studies will include experiments with primary patient samples, as well as in vivo trials combining Wee1 inhibition with Parp1/2 inhibition. These preliminary studies raise the possibility of rational combinations of targeted agents for leukemia in those for whom conventional chemotherapeutics may not be well tolerated. Disclosures No relevant conflicts of interest to declare.

scholarly journals Coagulation Factor XII Plays an Important Role in Procoagulant Activity of Apoptotic Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2457-2457
Author(s):  
Aizhen Yang ◽  
Yi Wu
Keyword(s):  
Knockout Mice ◽  
Conflicts Of Interest ◽  
Coagulation Factor ◽  
Annexin V ◽  
Procoagulant Activity ◽  
Contact System ◽  
Coagulation System ◽  
Factor Xii ◽  
Apoptotic Cells ◽  
Dependent Manner

Abstract Apoptosis can be induced in a variety of pathological disorders, including inflammation, autoimmune diseases, and chemotherapy. When cells undergo apoptosis, they express phosphatidylserine (PS) on cell membrane surface and thus become procoagulant. Although it has been known that the procoagulant activity of apoptotic cells are tightly associated with thrombotic disorders, such as atherothrombosis and Trousseau syndrome, the mechanisms by which apoptotic cells activate the coagulation system and enhance blood clotting are largely unknown. In this study we investigated which coagulation factor(s) is involved in this process. Using western blotting and chromogenic substrate assay, we found that incubation with apoptotic cells induced by Dexamethasone (DXMS), but not with viable cells, resulted in rapid cleavage and activation of FXII. Moreover, apoptotic cells-mediated FXII activation was significantly increased in the presence of prekallikrein (PK) and high molecular weight kininogen (HK), other two components of plasma contact system. However, incubation of apoptotic cells did not cause dramatic changes of other coagulation factors, suggesting a selective association of FXII activation with apoptotic cells. Activation of FXII by apoptotic cells was markedly inhibited by a specific anti-kallikrein antibody, indicating the activation of the contact system by apoprotic cells. Flow cytometric measurement showed that FXII bound to apoptotic cells in a concentration-dependent manner, which was inhibited by annexin V and PS liposome. A surface plasmon resonance assay showed a direct binding of FXII to PS (KD=3.9E-9 M). When challenged by apoptotic cells, clotting time of plasma from FXII-knockout mice was significantly prolonged, which was reversed by replenishment with human FXII. Moreover, an inhibitory anti-FXII antibody completely prevented apoptotic cells-induced intrinsic tenase complex formation. Consistently, apoptotic cells significantly increased thrombin production in normal plasma, which were attenuated by PS blocker annexin V, an inhibitory anti-FXII antibody, and the deficiency of FXII, respectively. Addition of human FXII to XII-deficient plasma recovered thrombin generation. As evaluated by ELISA, the levels of thrombin-antithrombin complex in circulation were significantly increased when apoptotic cells were intravenously injected into wild-type mice, but not in FXII-knockout mice. In conclusion, FXII plays an important role in apoptotic cells-mediated procoagulant activity. Disclosures No relevant conflicts of interest to declare.

scholarly journals Annexin V as a probe of aminophospholipid exposure and platelet membrane vesiculation: a flow cytometry study showing a role for free sulfhydryl groups

Blood ◽  
1993 ◽  
Vol 81 (10) ◽  
pp. 2554-2565 ◽  
Author(s):  
J Dachary-Prigent ◽  
JM Freyssinet ◽  
JM Pasquet ◽  
JC Carron ◽  
AT Nurden
Keyword(s):  
Flow Cytometry ◽  
Platelet Activation ◽  
Annexin V ◽  
Cytoskeletal Proteins ◽  
Procoagulant Activity ◽  
Ionophore A23187 ◽  
Calpain Activity ◽  
Platelet Surface ◽  
Reactive Agents ◽  
Platelet Secretion

Annexin V, a protein with a high affinity and a strict specificity for aminophospholipids at physiologic calcium concentrations, was used to probe platelet activation and the development of procoagulant activity. Platelet secretion was studied in parallel using VH10, a murine monoclonal antibody specific for GMP-140, an alpha-granule membrane glycoprotein. Both proteins were labeled with fluorescein isothiocyanate and platelet activation was assessed by flow cytometry. Microparticles, which are shed from the platelet surface and also support procoagulant activity, were distinguished from platelets according to their associated light scattering signal. The relative ability of different inducers to trigger exposure of the procoagulant surface and microparticle formation was: ionophore A23187 = thrombin plus collagen = collagen = thrombin. The density of aminophospholipid on microparticles was higher than on remnant platelets. Platelet activation by these agonists was accompanied by GMP-140 exposure, both on platelets and microparticles. Here, thrombin was the most efficient agonist. The mechanisms responsible for the above processes were investigated using E-64-d, a specific membrane-permeable inhibitor of Ca(2+)-activated protease (calpain); tetracaine, an activator of calpain; and N-ethylmaleimide and diamide, two sulfhydryl-reactive agents. These agents were added to platelets alone or before stimulation by agonists. Calpain activity was assessed by the hydrolysis of cytoskeletal proteins as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Results showed that calpain activity is not essential for aminophospholipid translocation or for secretion. In contrast, although sulfhydryl-reactive agents alone can trigger procoagulant activity, they inhibit microvesicle formation and platelet secretion induced by the above agonists, suggesting that different mechanisms account for these phenomena. The use of annexin V in flow cytometry is a rapid method to assess procoagulant activity in platelets and the loss of phospholipid asymmetry in cell membranes.

Elevated Procoagulant Activity Due to Phosphatidylserine Exposure on Red Blood Cells during Storage.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1996-1996
Author(s):  
Jin Zhou ◽  
Chengfang Lu ◽  
Hongjuan Yu ◽  
Jinxiao Hou ◽  
Shuchuan Liu ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Mononuclear Cells ◽  
Cell Types ◽  
Procoagulant Activity ◽  
Organ Injury ◽  
Enzymatic Assays ◽  
Healthy Donors ◽  
Coagulant Activity ◽  
Pathogenetic Factor ◽  
Relationship Of

Abstract Serious complications and mortality after cardiac surgery are increased when transfused red cells are stored for more than 2 weeks (Koch, N Engl J Med. 2008). However, the mechanism remains unclear and the relationship of procoagulant activity to PS exposure on banked RBCs has not been clarified, although prior studies suggest that banked cells went to apoptosis. Procoagulant activity of RBCs was measured in a modified prothrombin time in which RBCs replaced thromboplastin. The presence of PS in neutrophils and mononuclear cells from healthy donors and banked RBCs was investigated by flow-cytomety and confocal microscopy. The assembly of extrinsic tenase, intrinsic tenase, and prothrombinase complexes on these cells was studied using enzymatic assays employing plasma or purified proteins. Lactadherin, a glycoprotein, was utilized as a probe to track PS exposure and as an agent to block exposed PS. Coagulant activity increased approx 5-fold after cells were banked for 14 days and progressively increases. The percent of phosphatidylserine (PS)-positive cells increased from 7.4% on day 14 to 30% on day 42. RBCs with exposed PS were nearly absent in samples at 7 days but readily evident at 14 days on confocal microscopy. Banked RBCs exhibited patched, a rim, and diffuse-pattern that stained with lactadherin but neutrophils and MNC did not, indicating more PS exposure on banked RBCs from 7 days than the other two cell types. These data reveal a positive correlation between PCA and PS exposure on stored RBCs. Inhibition of PCA by lactadherin further confirms that PS supports the PCA in stored RBCs. Thus, we propose the possible role of PCA due to increased PS exposure on aged RBCs as a potential pathogenetic factor leading to organ injury, aggravated infections and finally higher mortality.

scholarly journals The Effect of the R882H DNMT3a Mutation on Megakaryocyte Cell Lines

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5186-5186
Author(s):  
Yang Lu ◽  
Dan Wang ◽  
Yifang Yuan ◽  
Li mei Chen ◽  
Yi ke Huang ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Platelet Count ◽  
Cell Lines ◽  
Colony Formation ◽  
Dna Methyltransferase ◽  
De Novo ◽  
Conflicts Of Interest ◽  
Causal Relation ◽  
Meta Analysis ◽  
Annexin V

Abstract Introduction DNA methyltransferase (DNMT) family play an important role in the development and growth of lives, encoding enzymes that catalyze the addition of a methyl group to the cytosine residue of CpG islands. With the increase in methylation, the downstream genes are often associated with reduced expression. In this family, DNMT3a occupies the essential position to implement the de novo methylation. Timothy J. Ley and many other scientists found that in M4 and M5 acute myeloid leukemia (AML), around 20% patients suffered from DNMT3a mutation (most are R882H mutation), always associating with adverse prognosis. But what's the reason for adverse prognosis? Additionally, our formal Meta analysis showed that the de novo AML patients with DNMT3a mutation have higher platelet counts, WBC and RBC counts. To shed some light on the possible causal relation between the increasing in platelet count and poor prognosis led by DNMT3a mutation, we transduced the MK cell lines with genes null-mCherry (null), DNMT3a wild type-mCherry (DNMT3aWT) and DNMT3a R882H mutation type-mCherry (DNMT3aMT) respectively, trying to figure out the possible role that the mutation plays in the megakaryopoiesis and thrombopoiesis. Also, we tested several drugs that may target the mutation. Methods The SFFV-null-IRES-mCherry, SFFV-DNMT3aWT-IRES-mCherry and SFFV-DNMT3aMT-IRES-mCherry plasmids were constructed by Dr. Qianli Jiang, modified from LEGO-iC plasmids. MK cell lines (chrf-288-11, meg-01) were flow-through transduced with the lentivirus produced by packaging plasmids and those above. All the fluorescence positive cells have been doubly sorted by flow cytometry. Cell ploidy was analyzed by flow cytometry using Propidium Iodide (PI); colony forming unit (CFU-MK) were enumerated 14d after being plated with TPO and IL-3; cell proliferation were tested by CCK-8; apoptosis was measured via flow cytometry with PI and Annexin V-FITC; CD41a and CD61 were tested with flow cytometry. The drug tests including Decitabine, Dasatinib and Rituximab were analyzed using CCK-8 test and cytomorphologic tests. Results With CCK8 test of chrf-288-11 and meg-01, DNMT3aMT proliferates faster than the null and DNMT3aWT (P<0.05, Fig.1). In CFU-MK, both cells lines showed that DNMT3a mutation promoted the colony formation (P<0.05). The CD41a percentage decreased from null to DNMT3aWT and DNMT3aMT (P<0.05) while the CD61percentage increased from null to DNMT3aWT and DNMT3aMT (P<0.05). Also, with morphologic analyses, DNMT3aMT in both cell lines maintain more mature stages. Cell ploidy test also demonstrated that cell lines with DNMT3a mutation contain more multiploids (P<0.05). Apoptosis test illustrated that DNMT3a mutation protect the cell lines from apoptosis (P<0.05). In the drug experiments, 1uM Decitabine could slow down the proliferation of 3 gene types of chrf-288-11 significantly (P<0.05). Dasatinib also posed a negative effect on the proliferation of 3 gene types of chrf-288-11 (P<0.05, Fig.2). In Rituximab experiment, we could find that interestingly, certain concentrations could speed up the proliferation of 3 gene types of chrf-288-11, while others not (P<0.05, Fig.3). Conclusion With all the above evidences, we can safely conclude that the megakaryocyte cell lines with DNMT3a mutation are associated with high-differentiation, high-colony formation and low-apoptosis, which could help us to understand the elevation of platelet count in AML patients with DNMT3a mutation. The anti-apoptosis and renewal ability of the cell lines with DNMT3a mutation may lead to a bad prognosis of these AML patients (with DNMT3a mutation). What's more, according to the drug experiment, we found in the both cell lines, DNMT3aWT and DNMT3aMT cells died significantly at even low concentration of decitabine. Dasatinib also slowed down the proliferation of 3 gene types of chrf-288-11, whether Dasatinib could lead to further treatment of such leukemia with DNMT3A mutation needs more research. Rituximab is helpful in the treatment against refractory thrombocytopenia. However, the mechanism hasn't been clarified. Interestingly, our results showed that, Rituximab may have a direct effect on MKs, giving a boost to the megakaryopoiesis and thrombopoiesis with certain concentration. Figure 1 Figure 1. Figure 2 Figure 2. Figure 3 Figure 3. Disclosures No relevant conflicts of interest to declare.

scholarly journals Comparison of Pharmacokinetics and Hemostatic Efficacy of Red Cell Microparticles (RMP) in Rabbits Using Different Infusion Regimens

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2811-2811 ◽  
Author(s):  
Wenche Jy ◽  
Carlos Bidot ◽  
Max E Johansen ◽  
Lawrence L Horstman ◽  
Rifat Pamukcu ◽  
...  
Keyword(s):  
Steady State ◽  
Continuous Infusion ◽  
Conflicts Of Interest ◽  
Isotonic Saline ◽  
Annexin V ◽  
Procoagulant Activity ◽  
Rapid Decline ◽  
Red Cell ◽  
Excessive Bleeding ◽  
Hemostatic Efficacy

Abstract INTRODUCTION: Excessive bleeding is a life-threatening challenge in many areas of medical practice, especially in surgical procedures and trauma care. Few treatment options are available to meet the challenge of preventing or treating excessive bleeding, and none of them is satisfactory. The efficacy of red cell microparticles (RMP) in reducing blood loss has been documented in rat and rabbit bleeding models, as summarized in a recent publication by Jy et al. [Thromb. & Haemost., 2013;110:751-60]. No adverse effects were noticed in short-term observation of either model with the effective dose used. The rate of clearance of RMP in vivo has not been analyzed systemically. The purpose of this study is to characterize the pharmacokinetics / rate of clearance of RMP in rabbit model by different infusion regimens and to establish the relationship between blood concentration and hemostatic efficacy of RMP. METHODS: (i) RMP were produced by high pressure extrusion method [Thromb. & Haemost., 2013; 110:751-60]. The resulting product was washed twice with isotonic saline, lyophilized, and stored at -80°C. (ii) Pharmacokinetics of RMP were measured using either bolus infusion of RMP (3x109 counts/kg) during 1 min., or a combination of bolus (1/3 of total RMP) followed by continuous infusion (2/3 of total RMP) for 30 min to the sedated non-bleeding rabbits. Blood samples (1 mL each) were collected at intervals: 5 min pre-injection, and at 1, 3, 5, 7.5, 10, 15, 20, 25, 30, 45, and 60 min post-injection. A sample size of 5 animals was used for each infusion regimen. Levels of RMP were assayed by flow cytometry with dual labeling: anti-CD235a-PE and Annexin V-FITC. The former is specific for human RMP and will not label rabbit RMP. The latter is not species sensitive and labels both human and rabbit MP. (iii)The procoagulant activity of RMP in rabbit blood was assayed by thromboelastogram (TEG). RESULTS:(1) BolusInfusion of RMP (3 x109 counts/kg) resulted in a rapid rise of RMP levels peaking at 1 min post-infusion followed by rapid decline to baseline by 10 – 15 min. The half-life (T1/2) in circulation was estimated to be ≈ 4 – 7 min. The peak RMP concentration reached 2.5 – 3.4 x107 counts/mL. (2) The 2 markers used yielded small but significantly different rates of clearance after reaching peak concentrations: the T1/2 by anti-CD235a was 6.2 ±1.0 min, and by annexin V was 4.4 ±0.7 min (p = 0.01). These results indicate that these two phenotypes of RMP were cleared by different mechanisms. RMP expressing phosphatidylserine (annexin V positive) seem to be cleared faster than those expressing CD235a. (3) Bolus followed by continuous infusion resulted in a smaller initial spike (0.7 – 1.0 x107 counts/mL) followed by a rapid decline to ≈25-30%, then steady rise over the course of 30 min. infusion, finally reaching a steady-state level of 0.4 -0.6 x107 counts/mL. RMP levels returned to baseline within 15 min after cessation of infusion. (4) The ex vivo TEG data revealed good correlation between rise and fall of circulating RMP and procoagulant activity. However, T1/2 for procoagulant activity was longer (7.4 ±1.2 min.) compared to T1/2of circulating RMP, whether measured by anti-CD235a or annexin V. On the other hand, bolus followed by continuous infusion resulted in steady elevation of procoagulant activity, with little fluctuation during the course of infusion. CONCLUSIONS: These data demonstrate that bolus followed by continuous infusion of RMP is capable of maintaining almost steady-state levels for extended periods, with concomitantly increased procoagulant activity. Accordingly, this regimen is expected to be the optimum clinical treatment for excessive bleeding. This work also demonstrates the existence of different rate of clearance for different phenotypes of RMP, suggesting that multiple mechanisms are involved in the clearance of RMP. Disclosures No relevant conflicts of interest to declare.

scholarly journals Annexin V as a probe of aminophospholipid exposure and platelet membrane vesiculation: a flow cytometry study showing a role for free sulfhydryl groups

Blood ◽  
1993 ◽  
Vol 81 (10) ◽  
pp. 2554-2565 ◽  
Author(s):  
J Dachary-Prigent ◽  
JM Freyssinet ◽  
JM Pasquet ◽  
JC Carron ◽  
AT Nurden
Keyword(s):  
Flow Cytometry ◽  
Platelet Activation ◽  
Annexin V ◽  
Cytoskeletal Proteins ◽  
Procoagulant Activity ◽  
Ionophore A23187 ◽  
Calpain Activity ◽  
Platelet Surface ◽  
Reactive Agents ◽  
Platelet Secretion

Abstract Annexin V, a protein with a high affinity and a strict specificity for aminophospholipids at physiologic calcium concentrations, was used to probe platelet activation and the development of procoagulant activity. Platelet secretion was studied in parallel using VH10, a murine monoclonal antibody specific for GMP-140, an alpha-granule membrane glycoprotein. Both proteins were labeled with fluorescein isothiocyanate and platelet activation was assessed by flow cytometry. Microparticles, which are shed from the platelet surface and also support procoagulant activity, were distinguished from platelets according to their associated light scattering signal. The relative ability of different inducers to trigger exposure of the procoagulant surface and microparticle formation was: ionophore A23187 = thrombin plus collagen = collagen = thrombin. The density of aminophospholipid on microparticles was higher than on remnant platelets. Platelet activation by these agonists was accompanied by GMP-140 exposure, both on platelets and microparticles. Here, thrombin was the most efficient agonist. The mechanisms responsible for the above processes were investigated using E-64-d, a specific membrane-permeable inhibitor of Ca(2+)-activated protease (calpain); tetracaine, an activator of calpain; and N-ethylmaleimide and diamide, two sulfhydryl-reactive agents. These agents were added to platelets alone or before stimulation by agonists. Calpain activity was assessed by the hydrolysis of cytoskeletal proteins as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Results showed that calpain activity is not essential for aminophospholipid translocation or for secretion. In contrast, although sulfhydryl-reactive agents alone can trigger procoagulant activity, they inhibit microvesicle formation and platelet secretion induced by the above agonists, suggesting that different mechanisms account for these phenomena. The use of annexin V in flow cytometry is a rapid method to assess procoagulant activity in platelets and the loss of phospholipid asymmetry in cell membranes.

Procoagulant Activity of T Lymphoblastoid Cells due to Exposure of Negatively Charged Phospholipid

2002 ◽  
Vol 87 (03) ◽  
pp. 442-449 ◽  
Author(s):  
P. Fábregas ◽  
M. Jardi ◽  
J. Cancelas ◽  
M. Rabaneda ◽  
J. Felez ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
High Capacity ◽  
Factor Xa ◽  
Annexin V ◽  
Procoagulant Activity ◽  
Binding Studies ◽  
Lymphoblastoid Cells ◽  
Established Cell Lines ◽  
Established Cell ◽  
High Concentrations

SummaryWe have characterised the Procoagulant activity (PCA) of six well-established cell lines by assays of tissue factor (TF), thrombin and FXa generation, flow cytometry using Annexin V, and by binding studies with Factors VIII and IXa.The monocytic (THP-1 & U937) and promyelocytic (NB4) cells expressed high concentrations of TF antigen and activity, whereas TF in the lymphocytic cells (Molt 4, Jurkat & Nalm 6) was very low or absent. However the T-lymphoblastoid cells (Molt 4 & Jurkat) promoted the generation of large amounts of thrombin despite their low TF content, and these cells were also the most active in supporting Factor Xa generation. Molt 4 cells bound Factors VIII and IXa with high capacity and their activity was inhibited by Annexin V. These results indicate that the PCA of T-lymphoblastoid lines is due to expression of negatively charged phospholipids. Flow cytometry studies showed Annexin V binding to the major population of nonapoptotic Molt 4 cells and the PCA of Molt 4 was not increased when apoptosis was induced by staurosporine, indicating that PCA is independent of apoptotic status.

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