scholarly journals Prothrombin activation in blood coagulation: the erythrocyte contribution to thrombin generation

Blood ◽  
2012 ◽  
Vol 120 (18) ◽  
pp. 3837-3845 ◽  
Author(s):  
Matthew F. Whelihan ◽  
Vicentios Zachary ◽  
Thomas Orfeo ◽  
Kenneth G. Mann
Keyword(s):  
Blood Cells ◽  
Thrombin Generation ◽  
Marked Decrease ◽  
Platelet Rich Plasma ◽  
Buffy Coat ◽  
Glycophorin A ◽  
Initiation Phase ◽  
Platelet Concentration ◽  
Corn Trypsin Inhibitor ◽  
Prothrombin Activation

Abstract Prothrombin activation can proceed through the intermediates meizothrombin or prethrombin-2. To assess the contributions that these 2 intermediates make to prothrombin activation in tissue factor (Tf)–activated blood, immunoassays were developed that measure the meizothrombin antithrombin (mTAT) and α-thrombin antithrombin (αTAT) complexes. We determined that Tf-activated blood produced both αTAT and mTAT. The presence of mTAT suggested that nonplatelet surfaces were contributing to approximately 35% of prothrombin activation. Corn trypsin inhibitor–treated blood was fractionated to yield red blood cells (RBCs), platelet-rich plasma (PRP), platelet-poor plasma (PPP), and buffy coat. Compared with blood, PRP reconstituted with PPP to a physiologic platelet concentration showed a 2-fold prolongation in the initiation phase and a marked decrease in the rate and extent of αTAT formation. Only the addition of RBCs to PRP was capable of normalizing αTAT generation. FACS on glycophorin A–positive cells showed that approximately 0.6% of the RBC population expresses phosphatidylserine and binds prothrombinase (FITC Xa·factor Va). These data indicate that RBCs participate in thrombin generation in Tf-activated blood, producing a membrane that supports prothrombin activation through the meizothrombin pathway.

Recombinant factor VIIa partially reverses the inhibitory effect of fondaparinux on thrombin generation after tissue factor activation in platelet rich plasma and whole blood

2004 ◽  
Vol 91 (03) ◽  
pp. 531-537 ◽  
Author(s):  
François Depasse ◽  
Tahar Chakroun ◽  
Meyer Samama ◽  
Ismail Elalamy ◽  
Grigoris Gerotziafas
Keyword(s):  
Whole Blood ◽  
Thrombin Generation ◽  
Factor Viia ◽  
Platelet Rich Plasma ◽  
Inhibitory Effect ◽  
Endogenous Thrombin Potential ◽  
Initiation Phase ◽  
Hemorrhagic Events ◽  
Kinetics Of ◽  
Prothrombin Activation

SummaryFondaparinux (Arixtra®), a specific AT-dependent FXa inhibitor, is effective and safe in the prevention and treatment of venous thromboembolism, but some major hemorrhagic events may occur. No specific antidote to fondaparinux has been proposed. Recombinant FVIIa (Novoseven®) could be used as an haemostatic treatment, but this option has not been well documented. We studied the effect of rFVIIa (1 µg/ml) on the inhibition of thrombin generation induced by fondaparinux (0.1µg/ml to 1 µg/ml). Coagulation was triggered in platelet rich plasma (PRP) or in whole blood by recalcification in the presence of diluted thromboplastin. In PRP thrombin generation was assessed using the thrombinoscope assay. In whole blood, prothrombin activation was assessed by measuring the kinetics of F1+2 formation using an ELISA assay. Fondaparinux at concentrations equal or greater than 0.5 µg/ml prolonged the initiation phase of thrombin generation, and reduced the velocity of prothrombin activation. It also decreased by 60% the endogenous thrombin potential. In the presence of fondaparinux (0.5 µg/ml to 1 µg/ml) rFVIIa accelerated the initiation phase of thrombin generation, but it did not significantly increase the endogenous thrombin potential. However, rFVIIa did not completely reverse the inhibitory effect of fondaparinux on the parameters of thrombin generation and prothrombin activation. This study shows that rFVIIa accelerates thrombin generation, but does not completely reverse the inhibitory effect of fondaparinux on thrombin generation. The potential clinical use of rFVIIa as haemostatic treatment of major bleedings related to fondaparinux has to be evaluated.

scholarly journals Added value of blood cells in thrombin generation testing

2021 ◽  
Author(s):  
Jun Wan ◽  
Joke Konings ◽  
Bas de Laat ◽  
Tilman Mathias Hackeng ◽  
Mark Roest
Keyword(s):  
Blood Cells ◽  
Thrombin Generation ◽  
Platelet Rich Plasma ◽  
Added Value ◽  
Coagulation System ◽  
Erythrocyte Membranes ◽  
Receptor Interaction ◽  
Contact Activation ◽  
Coagulation Management ◽  
Critical Determinant

The capacity of blood to form thrombin is a critical determinant of coagulability. Plasma thrombin generation (TG), a test that probes the capacity of plasma to form thrombin, has improved our knowledge of the coagulation system and shows promising utility in coagulation management. Although plasma TG gives comprehensive insights in the function of pro- and anticoagulation drivers, it does not measure the role of blood cells in TG. In this literature review, we discuss currently available continuous TG tests that can reflect the involvement of blood cells in coagulation, in particular the fluorogenic assays that allow continuous measurement in platelet rich plasma and whole blood. We also provide an overview about the influence of blood cells on blood coagulation, with emphasis on the direct influence of blood cells on TG. Platelets accelerate the initiation and velocity of thrombin generation by phosphatidylserine exposure, granule content release and surface receptor interaction with coagulation proteins. Erythrocytes are also major providers of phosphatidylserine and erythrocyte membranes trigger contact activation. Furthermore, leukocytes and cancer cells may be important players in cell-mediated coagulation, because under certain conditions, they express tissue factor, release procoagulant components and can induce platelet activation. We argue that testing TG in the presence of blood cells may be useful to distinguish blood cells-related coagulation disorders. However, it should also be noted that these blood cells-dependent TG assays are not clinically validated. Further standardization and validation studies are needed to explore their clinical usefulness.

scholarly journals A cross-sectional analysis of the effects of various centrifugation speeds and inclusion of the buffy coat in platelet-rich plasma preparation

2021 ◽  
Vol 0 ◽  
pp. 1-8
Author(s):  
K. Muthuprabakaran ◽  
Varadraj Vasant Pai ◽  
Suhail Ahmad ◽  
Pankaj Shukla
Keyword(s):  
Blood Cell ◽  
Platelet Rich Plasma ◽  
Autologous Blood ◽  
Buffy Coat ◽  
Cross Sectional ◽  
Medical Specialties ◽  
Yield Increase ◽  
Platelet Concentration ◽  
Sectional Analysis ◽  
The Ideal

Introduction: Platelet-rich plasma is an autologous blood preparation which is used in various medical specialties because of its regenerative properties. There is a wide variation in platelet-rich plasma preparation protocols and attaining the ideal platelet yield (>1 million platelets/μL) in a clinic setting can be challenging. We aimed at analyzing the centrifuge spin rates at which to attain an ideal platelet-rich plasma yield and also to study the effect of inclusion of the buffy coat after the first spin on the final platelet concentration in platelet-rich plasma. Methods: Seventy-five whole blood samples were obtained and divided into two groups – (1) leukocyte-rich platelet-rich plasma group and (2) leukocyte-poor platelet-rich plasma group. Samples in both groups were centrifuged using the dual spin method, at one of three centrifugation speed combinations (initial “soft” spin and second “hard” spin speeds, respectively): (1) 100 g/400 g, (2) 350 g/1350 g and (3) 900 g/1800 g. Platelet, red blood cell (RBC) and white blood cell (WBC) counts in both groups were compared. Results: The 100 g/400 g spin gave a high platelet yield (increase of 395.4 ± 111.1%) in the leukocyte-poor-platelet-rich plasma group, while in the leukocyte-rich platelet-rich plasma group both 100 g/400 g and 350 g/1350 g spins resulted in significantly higher yields with an increase of 691.5 ± 316.3% and 738.6 ± 193.3%, respectively. Limitations: The study was limited by a smaller sample size in the pure platelet-rich plasma (leukocyte-poor platelet-rich plasma) group. Conclusion: Ideal platelet yields can be achieved with both the 100 g/400 g as well as the 350 g/1350 g spins using the buffy coat inclusion method while the 100 g/400 g spin for “pure” platelet-rich plasma accomplishes a near-ideal platelet count with significantly reduced contamination with other cells.

scholarly journals The Effect of Autologous Platelet-Rich Plasma on Bronchial Stump Tissue Granulation after Pneumonectomy: Experimental Study

ISRN Surgery ◽  
2013 ◽  
Vol 2013 ◽  
pp. 1-6 ◽  
Author(s):  
Eleftherios Spartalis ◽  
Periklis Tomos ◽  
Petros Konofaos ◽  
Grigorios Karagkiouzis ◽  
Georgia Levidou ◽  
...  
Keyword(s):  
Platelet Rich Plasma ◽  
Healing Process ◽  
Bronchopleural Fistula ◽  
White Blood Cells ◽  
Buffy Coat ◽  
Optimum Number ◽  
Therapeutic Effects ◽  
Bronchial Stump ◽  
Platelet Concentration ◽  
Autologous Platelet Rich Plasma

Objectives. Recent advances in perioperative management, antibiotics, and surgical materials, including mechanical staplers, have decreased the operative risk of pulmonary resection. However, bronchopleural fistula can still occur in some instances, the occurrence often being lethal. This study investigated whether platelet-rich plasma (PRP) promotes granulation of the bronchial stump after pneumonectomy. Methods. Ten pigs were randomized into two groups: (A) control or non-PRP group (pneumonectomy) and (B) PRP group (pneumonectomy and PRP application). PRP was obtained by spinning down the animal’s own blood and collecting the buffy coat containing platelets and white blood cells. Results. Increased platelet concentration triggered the healing process. The percentage of granulation tissue formed at the stumps was significantly higher in the PRP group of animals. This observation was confirmed when statistical analysis using Mann-Whitney U test was performed (P=0.0268). Conclusions. PRP is easily produced with minimal basic equipment and is useful in accelerating granulation of the bronchial stump, although the timing and optimum number of applications in humans require further study. Autologous PRP is a safe, feasible, and reliable new healing promoter with potential therapeutic effects.

Platelets and Phospholipids in Tissue Factor-initiated Thrombin Generation

2001 ◽  
Vol 86 (08) ◽  
pp. 660-667 ◽  
Author(s):  
Saulius Butenas ◽  
Richard Branda ◽  
Cornelis van ’t Veer ◽  
Kevin Cawthern ◽  
Kenneth Mann
Keyword(s):  
Platelet Count ◽  
Tissue Factor ◽  
Whole Blood ◽  
Thrombin Generation ◽  
Phase 1 ◽  
Platelet Counts ◽  
Initiation Phase ◽  
Platelet Concentration ◽  
American Society

SummaryThe influence of platelets on tissue factor (TF)-initiated thrombin generation in a reconstituted model of blood coagulation and in whole blood was evaluated. No thrombin generation was observed over 15 min in the reconstituted model when either TF or platelets and phospholipids were omitted. At 25 pM TF, the rates of thrombin generation were platelet and PCPS concentration-dependent and achieved maximum (1.0 nM/s) in the physiological range of platelet concentration. Similar rates were achieved in the absence of platelets when 1-2 μM phospholipid was used. However, the maximum rates of thrombin generation (5.2-6.0 nM/s) and the shortest initiation phase (1 min) were attained between 25 and 100 μM phospholipid. In the reconstituted model, an increase in platelet concentration from 0.125 × 108/ml to 0.5 × 108/ml decreased the duration of the initiation phase (in the absence of phospholipids) from 4.3 min to 2 min. Further increases in platelet concentration did not affect this phase. Sequential whole blood studies were conducted in blood of a chemotherapy patient who developed reduced platelet counts. The TF (12.5 pM) initiated clotting of patient’s blood was accelerated from ~10 min to 5 min when the platelet concentration increased from 0.05 × 108/ml to 0.11 × 108/ml. Clotting times were essentially unchanged for platelet concentrations exceeding 0.5 × 108/ml (range 0.5-3.1 × 108/ml). Similarly, clotting of whole blood obtained from healthy volunteers was not affected by the platelet count, which varied from 1.5 × 108/ml to 3.1 × 108/ml (4.0 ± 0.5 min). The data obtained in both models are consistent with in vivo observations that clinical bleeding is most likely to occur at platelet counts <0.1 × 108/ml.Portions of this work were presented at the 40th Annual Meeting of the American Society of Hematology, December 4-8, 1998, Miami Beach, Florida (abstracts #140 and #738).

Plasma Microparticles Have Different Effects on Thrombin Generation in Platelet-Rich and Platelet-Poor Plasma.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1760-1760
Author(s):  
Gleb E. Ivanov ◽  
N. Macartney ◽  
E. Stephens ◽  
N. Bowen ◽  
S. Lees ◽  
...  
Keyword(s):  
Thrombin Generation ◽  
Platelet Rich Plasma ◽  
Maximal Velocity ◽  
Physiological Level ◽  
Wide Range ◽  
Coagulation Tests ◽  
Platelet Concentration ◽  
Vitro Effect ◽  
Free Plasma

Abstract Circulating peripheral blood microparticles (MPs) of various cell origin have been described and measured in physiological and a wide range of pathological conditions. MPs are likely to play a role in coagulation either by exposure of procoagulant phospholipids or expression of tissue factor (TF), but the degree of this contribution to global haemostasis is not yet clear. We studied thrombin generation (TG) parameters (lag, peak thrombin, initial velocity (Vini) and maximal velocity (Vmax) in platelet-free (PFP) and platelet-rich plasma (PRP) of normal volunteers (n=9) in presence of corn trypsin inhibitor, using calibrated automated thrombography (CAT). MP-rich plasma was prepared by ultracentrifugation of PFP and reconstitution of pelleted MPs in a reduced volume of autologous MP-free plasma. TG was also measured in MP-depleted supernatant and platelet-free plasma (PFP) filtered through 0.1 μm filter. In MP-rich plasma, triggered with 5pM TF, with no addition of exogenous phospholipids, we found significantly increased peak TG, compared with PFP and supernatant (70.8 +/− 6.3 vs 51.4 +/− 5.0 vs 28.4 +/− 2.2 nM/L thrombin, p=0.024 and p<0.0001 respectively). MP-rich fraction also produced raised Vini (10.3 +/− 0.9 vs 5.0 +/− 0.6 thrombin nM/L/min, p=0.019) and Vmax (18.3 +/− 2.4 vs 6.8 +/− 1.0 thrombin nM/L/min, p=0.004) compared with MP-depleted supernatant. Ultracentrifugation resulted in reduction of peak TG almost by half, compared with native PFP. The augmenting effect of MP-rich plasma on thrombin peak and velocity was shown to be abolished by filtration. In our experiments removal of MPs by filtration of PFP did not affect routine clinical coagulation tests, but resulted in a significant reduction of peak TG (from 51.4 +/− 5.0 to 23.9 +/− 1.4 thrombin nM, p=0.0002), Vini (from 10.2 +/− 0.4 to 5.6 +/− 0.6, p=0.02) and Vmax (from 15.2 +/− 1.8 to 5.9 +/− 0.2, p=0.02) as compared to PFP. In order to assess the contribution of MPs to TG in presence of platelets, MP-rich plasma was added to various dilutions of PRP, using low concentration of TF (0.5pM) as a trigger. Interestingly, addition of MP-rich fraction only marginally augmented PRP with a platelet concentration of 150x109/L, but the enhancement of peak and velocity of TG became more pronounced when platelet concentration was reduced to 1.5x109/L. In a separate set of experiments, we studied TG in PRP in which MP concentration was reduced by dilution with filtered MP-free plasma as compared to PRP diluted with MP-containing PFP. Reduction in PRP MP content did not lead to a significant decrease in TG even at a low platelet concentration (1.5x109/L), when MP concentration was reduced to about 100 times below the physiological level. Our results indicate that MPs contained in PFP of normal donors significantly affect thrombin generation peak and velocity when compared to PFP in which MPs were eliminated by either ultracentifugation or filtration. The in vitro effect of an increased number of MPs on TG is less noticeable in presence of near-physiological platelet count, but contribution of MPs to TG at low platelet concentrations may potentially protect from bleeding in thrombocytopenic states and explain differences in bleeding phenotype. CAT measurement of TG in MP-rich vs MP-poor plasma could serve as a useful tool in assessing these differences.

Assessment of the Clinical Benefit of Prophylactic Platelet Transfusion in Thrombocytopenia Using Novel Methodology To Measure Thrombus Formation and Thrombin Generation.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 949-949
Author(s):  
Sandra Cauwenberghs ◽  
Marion A.H. Feijge ◽  
Johan W.M. Heemskerk ◽  
Elisabeth van Pampus ◽  
Joyce Curvers
Keyword(s):  
Thrombin Generation ◽  
Platelet Rich Plasma ◽  
Thrombus Formation ◽  
Synthetic Medium ◽  
Blood Platelet ◽  
Severe Thrombocytopenia ◽  
Experimental Conditions ◽  
Platelet Function Test ◽  
Hemostatic Activity ◽  
Platelet Concentration

Abstract Patients developing severe thrombocytopenia during chemotherapy treatment are prophylactically transfused with platelets. Two novel methods were developed for measuring improved hemostasis in thrombocytopenic patients and to identify patients with aberrant responsiveness. In whole blood, platelet adhesion and thrombus formation on collagen was measured under flow. In platelet-rich plasma, platelet-dependent coagulation was assayed by automated measurement of thrombin generation. Thirty-eight thrombocytopenic patients were transfused with platelets stored in plasma or synthetic medium (PASII), and the transfusion effect on hemostatic activity was evaluated. Experimental conditions were established, where the outcome of flow and thrombin generation tests linearly increased with the platelet concentration (R2=0.98, p=0.001 and R2=0.98, p=0.008 respectively), and informed on the activation properties of platelets. In 35 out of 38 patients, transfusion with platelets in plasma or synthetic medium resulted in increased adhesion and thrombus formation under flow, and in increased platelet-dependent coagulation. The increase in platelet count after transfusion predicted 57% (P=0.001) of the improvement in function. Transfusion with platelets in plasma or synthetic medium increased the hemostatic activity with 0.63±0.30% and 0.68±0.48%, as determined with either platelet function test, with subtle differences between the two storage media. In acute graft-versus-host-disease, platelet-dependent coagulation was higher than in other patients, while thrombus formation was normal. In conclusion, altered thrombus formation and/or aberrant coagulation at low platelet counts can be detected with these novel developed and validated techniques.

Erythrocyte Participation in Thrombin Generation

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 374-374
Author(s):  
Matthew Whelihan ◽  
Kenneth G. Mann
Keyword(s):  
Board Of Directors ◽  
Trypsin Inhibitor ◽  
Whole Blood ◽  
Thrombin Generation ◽  
Maximum Level ◽  
Lag Phase ◽  
Advisory Committees ◽  
Contact Pathway ◽  
Corn Trypsin Inhibitor ◽  
Prothrombin Activation

Abstract Abstract 374 Introduction: The potential contributions of erythrocytes (RBCs) to coagulation biochemistry have been controversial. We evaluated the potential for RBCs to participate in thrombin generation using minimally altered whole blood. Methods: Platelet poor plasma (PPP), platelet rich plasma (PRP) adjusted to a physiologic concentration with PPP, washed RBCs (PLTs <0.5%) and washed PLTs were prepared from contact pathway inhibited (corn trypsin inhibitor, CTI) whole blood in the absence of other anticoagulants. RBCs and PLTs were evaluated for phosphatidylserine (PS) exposure by FACS using FITC-labeled bovine lactadherin and their ability to support prothrombinase (1.4μM prothrombin, 20nM factor Va, 200pM factor Xa (FXa), 3.4μM antithrombin (AT) at physiologic concentrations of RBCs and PLTs). CTI whole blood and prepared sub fractions (PRP, PPP, PRP+RBC, PPP+RBC) were subjected to a 5pM tissue factor (TF) stimulus and samples analyzed by Western blotting and α-thrombin (αIIa) antithrombin (αTAT) ELISA (Rand et al. Blood, 1996). Results: CTI whole blood (N=3 donors mean±SD) clotted in 4±1.5 min whereas the matching PRP clotted in 8±2 min. In αTAT ELISA analyses, PRP showed a corresponding increase in lag phase and a 50% decrease in the maximum rate (26±9nM/min vs 58±7nM/min) and extent (263±62nM vs 476±86nM) of αTAT formation compared to whole blood. When PRP was reconstituted with physiologic levels of washed RBCs, the rate and extent of αTAT formation as well as the lag phase were restored to that observed in CTI whole blood. Addition of buffy coat to PRP, to test white blood cell contributions, had no effects on any of these parameters. Western blotting showed a significant decrease in prothrombin consumption in the PRP experiments compared to whole blood and PRP with washed RBCs. When RBCs were added to PPP (N=1), there was no significant prolongation of the lag phase and the rate of αTAT formation (21nM/min) was half of that seen in the corresponding whole blood (41nM/min). However, the maximum αTAT generated in RBCs+PPP (267nM) vs whole blood (343nM) differed by only 22%. Collectively, these data indicate a significant role for RBCs in the propagation of thrombin generation in CTI whole blood. FACS analysis of PS exposure on RBCs showed the following: untreated RBCs showed minimal binding to lactadherin (1.3%) compared to the positive control (99.7%; RBCs treated with 10mM N-ethylmaleimide (NEM) and 4μM ionophore A23187); pretreatment of the RBC population with 10nM αIIa or FXa showed a 5-fold increase in lactadherin binding over the untreated control indicating the presence of surface conditions capable of supporting prothrombinase. This proportion (5%) of PS-expressing RBCs represents a similar proportion of PLTs to RBCs (4%) in whole blood. Prothrombin activation (N=2)(1.4μM II, 20nM FVa, 200pM FXa, 3.4μM AT) on untreated RBCs exhibited a 3.5±0.5 minute lag phase followed by αTAT production which reached a rate of 18nM/min and maximum level of 180nM. In experiments performed in the absence of AT, pretreatment of RBCs with either 10nM αIIa or FXa shortened the lag phase by 1±0.25min. PAR-1 (TFLLRN) activated PLTs or activated PLTs+RBCs showed no lag phase, and no difference in the rate of αTAT production (25nM/min) or maximum level (200nM) of αTAT generated. Conclusions: In contact pathway-inhibited whole blood initiated with TF, RBCs appear essential for normal thrombin generation. Our approach rapidly fractionates CTI blood in the absence of other anticoagulants and yields populations of minimally altered RBCs that when subjected to a Tf stimulus produce thrombin more rapidly than PRP. Physiologic levels of washed RBCs pretreated with αIIa or FXa support a level of prothrombin activation similar to that observed with washed activated PLTs. These findings suggest that RBCs participate in thrombin generation and produce a PS-equivalent membrane when treated with αIIa or FXa. Disclosures: Mann: Haematologic Technologies: Chairman of the Board, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; corn trypsin inhibitor: Patents & Royalties; NIH, DOD, Baxter: Research Funding; Merck, Daiichi Sankyo, Baxter, GTI: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Contribution of Erythrocytes to Thrombin Generation in Whole Blood

1999 ◽  
Vol 81 (03) ◽  
pp. 400-406 ◽  
Author(s):  
V. Peyrou ◽  
J. C Lormeau ◽  
J. P. Hérault ◽  
C. Gaich ◽  
A. M. Pfliegger ◽  
...  
Keyword(s):  
Thrombin Generation ◽  
Antithrombin Iii ◽  
Platelet Rich Plasma ◽  
Factor Xa ◽  
Annexin V ◽  
Lag Time ◽  
Glycophorin A ◽  
Response Curves ◽  
First Time ◽  
Prothrombinase Activity

SummaryThrombin generation (TG) initiated by diluted tissue-factor was investigated in whole human blood, in platelet-rich plasma (PRP), in platelet-poor plasma (PPP), and in PPP supplemented with red blood cells (RBCs). TG was characterized by the lag time preceding the thrombin burst and by the endogenous thrombin potential (ETP). RBCs at normal haematocrit were found to influence the lag time to the same extent as platelets. When TG was carried out in PRP or in PPP + RBCs, both the ETP and lag time were dependent on the platelet count or on the haematocrit, but the shapes of the dose-response curves were different. The inhibition of TG in PPP+ RBCs by two direct thrombin and factor Xa inhibitors: hirudin and DX 9065A, and two antithrombin III (AT)-dependent anticoagulants: heparin and SR 90107A was found to be similar to that previously described in PPP and in PRP: hirudin and DX 9065A only delayed TG whereas heparin and SR 90107A both delayed and decreased TG. FACscan analysis following labelling with FITC-annexin V or with phycoerythrin-labelled anti-glycophorin A of samples taken in the course of TG initiated in PPP + RBCs showed that no significant haemolysis occurred and revealed that 0.51 ± 0.075% (mean ± sem, n = 3) of RBCs steadily exposed procoagulant phospholipids on their outer surface throughout the TG course. Furthermore, incubation of factors Xa and Va with washed RBCs sampled during TG in PPP +RBCs resulted in a significant and constant prothrombinase activity.Taken together, these data show for the first time that normal RBCs may participate in the haemostatic process through exposure of procoagulant phospholipids.

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