Procoagulant Phospholipid Activity Research Articles

Procoagulant Phospholipid Activity and MPV Values in Acute Ischemic Stroke

Ischemic stroke can be caused by atherothrombosis or embolism. Atherothrombosis occurs due to the rupture of an atherosclerotic plaque causing platelet activation. There are several markers of platelet activation, including platelet microparticles. The levels of platelet microparticles can be measured by examining procoagulant phospholipid (PPL) activity. It is not yet known exactly what activates thrombopoiesis, which can be assessed by an increase in Mean Platelet Volume (MPV). This study aimed to know whether there is an increase in PPL activity and MPV values in acute ischemic stroke, whether platelet activation is influenced by differences in ischemic stroke subtypes, and whether there is a relationship between PPL activity and MPV values in acute ischemic stroke. The study design was cross-sectional and involved 60 subjects. PPL activity in acute ischemic stroke (65.14±13.35 seconds) tends to be higher (shorter clotting time) than in healthy individuals (68.59±8.56 seconds), however, this difference was not statistically and clinically significant. The MPV value in acute ischemic stroke (9.83±0.72 fL) compared to healthy individuals (9.65±0.86 fL) however this difference was not statistically significant. PPL activity in the SAO subtype (61.66±1.31 seconds) tends to be higher than LAA (68.62±14.57 seconds), however, this difference was also not statistically significant. There was a weak correlation between PPL activity (seconds) and MPV value (fL) in acute ischemic stroke (r =0.34, p-value=0.03).

Rosuvastatin treatment decreases plasma procoagulant phospholipid activity after a VTE: A randomized controlled trial

BackgroundVenous thromboembolism (VTE) is a frequent cardiovascular disease with severe complications, including recurrence and death. There is a great need for alternative prophylactic treatment options as anticoagulation is accompanied by increased bleeding risk. Statins are reported to reduce the risk of incident and recurrent VTE, but the mechanisms are elusive. Procoagulant phospholipids (PPL), and phosphatidylserine in particular, are crucial for efficient coagulation activation, but no studies have investigated the effect of statin treatment on plasma PPL activity. ObjectivesTo investigate the impact of rosuvastatin treatment on plasma PPL activity and levels of extracellular vesicles (EVs). Patients/MethodsPatients with a history of VTE (≥18 years) allowed to stop anticoagulant treatment were randomized to either 20 mg/day of rosuvastatin treatment or no treatment for 28 days in the Statins Reduce Thrombophilia (NCT01613794) trial. Plasma samples were collected at baseline and study end. PPL activity was measured in samples from 245 participants using a factor Xa‐dependent clotting assay and EV levels by flow cytometry. ResultsRosuvastatin treatment yielded an overall 22% (95% confidence interval [CI] −38.2 to −5.8) reduction in PPL activity, and 37% (95% CI −62.9 to −11.2) reduction in PPL activity in participants with a history of pulmonary embolism. The effect of rosuvastatin on plasma PPL activity was not explained by changes in total cholesterol nor change in levels of total‐ or platelet‐derived EVs. ConclusionsRosuvastatin treatment caused a substantial decrease in plasma PPL activity, suggesting that a PPL‐dependent attenuation of coagulation activation may contribute to a reduced VTE risk following statin treatment.

Prothrombotic abnormalities in patients with multiple myeloma and monoclonal gammopathy of undetermined significance

BackgroundMultiple myeloma (MM) and its precursor condition, monoclonal gammopathy of undetermined significance (MGUS) have an increased risk of thrombotic events, especially during anti-myeloma treatment. Many different underlying causes for this hypercoagulability have been suggested, but current techniques to identify abnormalities in these patients are sparse and inefficient. The aim of this study was to assess the hypercoagulability in MGUS and MM patients through various coagulation analyses and identify changes in the MM patients throughout their treatment regimen. Materials and methodsPlatelet-free plasma from 38 MM patients, 19 MGUS patients and 34 healthy controls were tested for hypercoagulability using calibrated automated thrombogram, a procoagulant phospholipid assay, a microvesicle-associated (MV) tissue factor (TF) assay, and a cell-free deoxyribonucleic acid (cf-DNA) assay as a surrogate measurement for neutrophil extracellular traps (NETs). ResultsMGUS and MM patients both had elevated thrombin generation and procoagulant phospholipid activity in comparison to the control subjects. MM, and partly MGUS, showed increased MV-TF activity, however, only MM had increased levels of the cf-DNA. ConclusionsHere we demonstrated that hypercoagulability was present in patients with MGUS and MM through increased thrombin generation, possibly due to higher TF and procoagulant phospholipids (PPL) activity. This may be associated to MVs and, for MM patients, be attributed to procoagulant NETs activity; however, this remains to be determined.

Differences in the Coagulation Profile in Women with Mild and Severe Preeclampsia

Introduction:Different coagulations abnormalities have been referred in women with early oncet preeclampsia (EOP), but there are only few studies comparing those changes regarding to the severity of the disease. Aim:In this study we aimed to investigate the differences between the coagulation profile in women with mild and severe preeclampsia. Methods:This is an observational retrospective case-control study. Plasma samples were collected from 84 women divided into three groups, the healthy pregnant (HP) group (n=35), the mild preeclampsia (MP) group (n=34) and the severe preeclampsia (SP) group (n=15). The study population general characteristics are shown in Table 1. We studied the following biomarkers of hypercoagulability and endothelial cell activation: Tissue factor activity (TFa), Procoagulant phospholipid activity (PPL), Protein S, D-Dimers, Antithrombin, thrombomodulin, TFPI levels. All women were assessed with classic coagulation tests (aPTT and PT) fibrinogen levels and hemogram. Statistical analysis was performed using the PASW Statistics 17.0.2 (SPSS Inc.) for Windows. Results:Women with preeclampsia - mild or severe- showed significant increase of TFPI, TFa and TMa levels as compared to healthy pregnant women. No significant difference of TFPI, TFa was observed between MP and SP groups. In contrast, TMa levels were significantly increased in SP as compared to MP group. The ratio TFa/TFPI was also lower in SP as compared to MP-group. Women in MP or SP group had similarly shorter PPL clotting time as compared to HP group. D-dimer levels were increased in women with preeclampsia as compared to the HP group. D-Dimer levels were significantly higher in SP as compared to MP group. The levels of free PS activity in HP as well as MP and SP groups were lower than normal range in non-pregnant women and the value in MP was significantly lower than that of the HP or SP. Fibrinogen levels were not significantly different in the three studied groups of pregnant women. Prothrombin time was found to be increased in cases as compared to that in the controls. The mean value of prothrombin time in mild preeclampsia was 13.24±0.80 seconds and in severe preeclampsia it was seconds 14.77±0.96 and in pregnant controls 12.23±0.59 seconds (p<0.05 and p<0.001 respectively). The mean prothrombin time was found to increase with increasing severity of disease (p<0.001). The mean activated partial thromboplastin time were increased in mild preeclampsia and was 32.64±1.83 seconds and in severe preeclampsia it was 35.59±1.53 seconds and in pregnant controls 29.53±1.62 seconds (p<0.001). The activated partial thromboplastin time was found to increase with increasing severity of disease (p<0.001). The antithrombin III decreased in severe SP and MP or compared to pregnant controls (76.33±4.32 and 88.06±9.68 versus 95.40±0.36 respectively; p<0.001). This decrease is more pronounced in SP compared to MP (p<0.001). Conclusions:Preeclampsia is associated with endothelial cell activation as documented by the increase of TFa, soluble TM levels and TFPI levels in plasma. Release of soluble thrombomoduline and TFPI rather than TFa by endothelial cells appear to be related with degree of preeclampsia severity. Women with preeclampsia showed marked decrease of PPL clotting time indicating enhanced platelet activation that was independent of the severity of preeclampsia. In contrast, women with severe preeclampsia showed signs of enhanced hypercoagulability documented by the increase of D-dimer levels consumption of natural coagulation inhibitors and particularly of AT. This phenomenon tended to be reflected on the prolongation of PT and aPTT in women with severe preeclampsia. Disclosures No relevant conflicts of interest to declare.

VIIaAT complexes, procoagulant phospholipids, and thrombin generation during postprandial lipemia.

Factor VII activation occurs postprandially. A proportion of activated factor VII (VIIa) circulates in complex with antithrombin (VIIaAT). Our primary objective was to assess the effects of postprandial lipemia on circulating VIIaAT, procoagulant phospholipid (PPL) activity, and thrombin generation. Plasma samples from postmyocardial infarction patients (n=40) and controls (n=39) were taken before and at 3 and 6hours during a standardized oral fat tolerance test (OFTT). Fasting PPL activity measurements were also made in a second cohort of 108 postinfarction patients and 109 controls. VIIaAT was analyzed with the Asserachrom VIIaAT ELISA, PPL activity with the STA-Procoag-PPL kit, and thrombin generation with calibrated automated thrombogram with PRP-Reagent as trigger (all Diagnostica Stago products). Postprandially, VIIaAT increased in all samples without significant case-control differences in the overall response during the OFTT. Thrombin generation measures peak height and velocity, and PPL activity, were marginally affected by the test meal in the controls. Levels of all patient baseline measures were significantly different from controls, indicating a more hypercoagulable state, and these differences were maintained throughout the OFTT. Fasting samples from cases showed higher PPL activity than control samples. Viewing VIIaAT quantitation as a surrogate for TF activity measurement, postprandial increase in VIIaAT may reflect a mechanism that adds to the cardiovascular risk associated with postprandial lipemia. On the other hand, the impact of postprandial lipemia on PPL activity and thrombin generation seems to be minor.

Changes in the pattern of plasma extracellular vesicles after severe trauma.

BackgroundExtracellular vesicles (EV) released into the circulation after traumatic injury may influence complications. We thus evaluated the numbers of EV in plasma over 28 days after trauma and evaluated their pro-coagulant and inflammatory effects.Methods and findings37 patients suffering trauma with an injury severity score >15 were studied along with 24 healthy controls. Plasma samples were isolated by double centrifugation (2000g 20min; 13000g 2min) from blood collected from within an hour up to 28 days after injury. Plasma EV were counted and sized using nanoparticle tracking analysis (NTA); counts and cellular origins were also determined by flow cytometry (FC) using cell-specific markers. Functional effects were tested in a procoagulant phospholipid assay and in flow-based, leukocyte adhesion assay after endothelial cells (EC) were treated with EV.We found that EV concentrations measured by NTA were significantly increased in trauma patients compared to healthy controls, and remained elevated over days. In addition, or FC showed that patients with trauma had higher numbers of EV derived from platelets (CD41+), leukocytes (CD45+) and endothelial EC (CD144+). The increases were evident throughout the 28-day follow-up. However, the FC count represented <1% of the count detected by NTA, and only 1–2% of EV identified using NTA had a diameter >400nm. The procoagulant phospholipid activity assay showed that patient plasma accelerated coagulation on day 1 and day 3 after trauma, with coagulation times correlated with EV counts. Furthermore, treatment of EC for 24 hours with plasma containing EV tended to increase the recruitment of peripheral flowing blood mononuclear cells.ConclusionsEV counted by FC represent a small sub-population of the total load detected by NTA. Both methods however indicate a significant increase in plasma EV after severe traumatic injury that have pro-coagulant and pro-inflammatory effects that may influence outcomes.

Procoagulant Phospholipid Activity, Whole Blood Thromboelastography and Thrombin Generation Assay to Detect Hypercoagulability in Thalassemic Children

INTRODUCTIONLife expectancy of thalassemia patients has markedly increased over the last few decades. Nevertheless patients suffer from many complications of this congenital chronic disease.The presence of a highincidence of thrombotic events has led to the identification of a hypercoagulable state in these patients.The thrombotic risk is higher in β-thalassemia intermedia and splenectomized patients. The mechanisms responsible for the increased thrombotic risk are still unclear. Several factors that contribute to the hypercoagulable state in patients with thalassemia have been identified: chronic platelet activation, abnormal red blood cells, microparticles,iron overload, endothelial damage, splenectomy, decreased levels of anticoagulant factors and presence of prothrombotic mutations. We aimed to assess hypercoagulability in children with β-thalassemia. DESIGN AND METHODSSixty eight thalassemia major and 42 thalassemia intermedia patients included our study. The control group consisted of 41 age and sex matched healthy children. Demographic data of patients were recorded from their medical records. None of the thalassemic patients have thrombosis before. To evaluate the relative role of microparticles, blood cells and plasma: coagulation tests (prothrombin time, activated prothrombin time, fibrinogen and d-dimer), serum coagulation factor levels (factor II, V, VII, VIII, IX, X, von willebrand factor, protein C, protein S, antithrombin III), procoagulant phospholipid activity and thrombin generation assay were studied from plasma, thromboelastography from whole blood.The main component of microparticles are negative anionic phospholipids.Procoagulant phospholipid activity is a functional analyse to detect microparticles.Thromboelastography measures indices of the viscoelastic properties of whole blood after activation of coagulation and the thrombin generation assay measures the actual thrombin concentrations before and after the clot is formed. RESULTSThe median age is 144 months ( 11-236 months)in thalassemia major and 142 months (72-202 months) in thalassemia intermedia patients. Plasma factor II, factor V, factor IX, factor X and protein C levels were significantly lower in thalassemia major and intermedia patients than control subjects. Plasma phospholipid activity and whole blood thromboelastography parameters were all consistent with hypercoagulability in thalassemic patients, especially in splenectomized patients. Endogenous thrombin potential (area under the curve in thrombin generation assay) was significantly lower in thalassemic patients than control subjects and in non-splenectomized patients than splenectomized patients contrary to expectations. CONCLUSIONSThe hypercoagulability in thalassemic patients especially in splenectomized patients can be determined with procoagulant microparticle activity and whole blood thromboelastography but not with thrombin generation assay in platelet poor plasma. These findings showthat blood cells and/or platelets may be more important determinants of thrombotic risk rather than plasma abnormalities in thalassemic patients. DisclosuresNo relevant conflicts of interest to declare.

Phospholipid‐dependent procoagulant activity is highly expressed by circulating microparticles in patients with essential thrombocythemia

This study evaluates the functional procoagulant features of plasma microparticle (MP) to explore the MP contribution to the hypercoagulable state of patients with essential thrombocythemia (ET). Platelet-free plasma samples were obtained from 73 ET patients (37 positive for the JAK2V617F mutation) and 72 control subjects. The calibrated automated thrombogram (CAT) was performed in plasma samples to determine thrombin generation of MP-associated tissue factor (TF) and procoagulant phospholipid (PPL) activity, and the STA Procoag PPL assay to measure MP-PPL activity only. Both thrombin generation and PPL procoagulant activities were found significantly elevated in ET patients compared to controls, and were associated to significantly higher levels of TF antigen and FVIIa/AT complex. Thrombin generation was significantly greater in JAK2-V617F positive compared to JAK2-V617F negative patients and normal subjects. Significant correlations were found between the PPL-assay and the different parameters of the CAT assay. No difference was seen between the thrombosis and no thrombosis group. Prospective studies are needed to test whether MP-associated thrombin generation and procoagulant activity may predict for thrombosis in these patients.

The microparticle‐specific procoagulant phospholipid activity changes with age

The microparticle‐specific procoagulant phospholipid activity changes with age

Relationship between short activated partial thromboplastin times, thrombin generation, procoagulant factors and procoagulant phospholipid activity

Short activated partial thromboplastin times (APTTs) are associated with thrombosis. However, what short APTTs actually represent in terms of possible mechanistic pathways is not well characterized. We have assessed thrombin generation as compared with levels of procoagulant factor (fibrinogen, V, VIII, IX, XI and XII) activities, von Willebrand factor level and activity using collagen binding, as well as procoagulant phospholipid activity, in 113 consecutive samples exhibiting a short APTT compared with an equal number of age-matched and sex-matched samples yielding a normal APTT. We found a significant difference in peak thrombin generation, velocity index and area under the curve between the two groups, and that thrombin generation markers correlated with the APTT, procoagulant phospholipid activity and several procoagulant clotting factors. We conclude that short APTTs represent a procoagulant milieu, as represented by heightened thrombin generation and several other heightened procoagulant activities, which may help explain the association with thrombosis.

Comparison of Clot-Based vs Chromogenic Factor Xa Procoagulant Phospholipid Activity Assays

We compared 2 commercial plasma procoagulant phospholipid activity (PPA) assays, chromogenic, using bound annexin V to capture phosphatidylserine-containing microparticles, and clot-based. In both, anionic phospholipids accelerated activation of prothrombin by factor Xa. PPA levels were lower in the chromogenic vs the clot-based assay, with poor correlation between methods: normal samples, mean ± SD, 27 ± 17 vs 590 ± 414 nmol/L (n = 24; r(2) = 0.29) and patient samples, mean ± SD, 45 ± 44 vs 401 ± 330 nmol/L (n = 51; r(2) = 0.26). Recovery of phosphatidylserine added to normal, heparinized, and warfarin plasma samples averaged 109% ± 39% using the chromogenic assay but was higher and more varied (mean ± SD, 176% ± 59%) in the clot-based assay. Lupus anticoagulants caused low recovery in both assays. Removal of microparticles by 0.22-μm filtration reduced PPA by 91% in the clot-based and 65% in the chromogenic assay. The clot-based assay showed higher correlation (r(2) = 0.82 vs 0.23) with flow cytometric platelet microparticle counts. The 2 assays measure different aspects of PPA in plasma, with the chromogenic assay primarily measuring smaller microparticles.

Procoagulant activity, but not number, of microparticles increases with age and in individuals after a single venous thromboembolism

The Calibrated Automated Thrombogram (CAT), a plate-based assay that measures thrombin generation and inhibition in plasma samples, is modified to measure the procoagulant activity of phospholipid associated with plasma microparticles (MP). The assay uses a tissue factor trigger without addition of 4 μM exogenous phospholipid (PL) used in the standard CAT. Calibrated with 4:1 posphatidylcholine- phosphatidylserine (PCPS) liposomes, the assay defines a median of 40 nM procoagulant phospholipid (PPL) equivalents in plasma containing MPs from 50 normal donors, with a range from 20 – 200 nM. Like the standard CAT, the modified assay detected no difference in plasma from 36 individuals with a history of a single episode of venous thromboembolism. However the male cases had double the PPL activity, as measured by rate of thrombin generation, of females; and there was a significant correlation among cases of increased thrombin generation with age. In contrast, there were no gender disparities or age correlations among control plasmas. The findings suggest that procoagulant activity of plasma microparticles, facilitated by a simplified, one-stage plate-based assay, offer a promising avenue of investigation of mechanisms and management of venous thromboembolic disorders.

The majority of circulating platelet-derived microparticles fail to bind annexin V, lack phospholipid-dependent procoagulant activity and demonstrate greater expression of glycoprotein Ib

It has been widely accepted that microparticles expose phosphatidylserine which in turn binds annexin V. It was the objective of this study to compare the antigenic characteristics and phospholipid-dependent procoagulant activity of annexin V positive and -negative subpopulations of platelet-derived microparticles. Annexin V positive and -negative microparticles were identified and characterised using flow cytometry and procoagulant activity was measured by a phospholipid-dependent assay (XACT). In unstimulated platelet-poor plasma, 80% of platelet-derived microparticles failed to bind annexin V. Varying the assay constituents (buffer, calcium and annexin V concentration) did not alter annexin V binding. The proportion of microparticles that bound annexin V was dependent upon the agonist, with physiological agonists such as collagen resulting in fewer annexin V binding microparticles than non-physiological agonists such as ionophore. CD42b (glycoprotein Ib) expression was significantly decreased and CD62p and CD63 expression were significantly increased in annexin V positive compared to annexin V negative subpopulations. There was no significant difference in CD41, CD61, CD42a and CD40L expression between annexin V positive and -negative subpopulations. A significant correlation between annexin V binding and XACT was found (p=0.033). Annexin V inhibited greater than 95% of phospholipid activity, suggesting that annexin V binding was a true reflection of procoagulant activity. The majority of platelet-derived microparticles in unstimulated plasma failed to bind annexin V and showed significantly increased levels of CD42b compared to annexin V positive events. Phospholipid-dependent procoagulant activity is limited to the annexin V positive subpopulation and is agonist-dependent. The significance of annexin V negative microparticles is unclear, however, it is possible that they possess other activities aside from procoagulant phospholipid activity.

Detection of the procoagulant activity of microparticle-associated phosphatidylserine using XACT

One of the mechanisms by which platelet-derived microparticles elicit procoagulant activity is by an increased exposure of phosphatidylserine on their surface. We have previously demonstrated the utility of an activated factor X-based assay for the detection of procoagulant phospholipid activity [Xa clotting time (XACT)]. The objective of this study was to further characterize the specificity of the XACT to detect microparticle-associated procoagulant phospholipid activity. XACT testing for procoagulant phospholipid was measured using an ST4 machine and microparticle counting was performed using flow cytometry for Annexin V binding. Plasma microparticle counts were significantly correlated to XACT times (P = 0.0001). The XACT assay was insensitive to tissue factor, whereas the addition of microparticles to a whole blood sample shortened XACT times. Procoagulant phospholipid activity could be detected in both citrate and EDTA anticoagulated samples; however, XACT times and microparticle counts were more stable in EDTA anticoagulated samples over a 60 min period. The procoagulant phospholipid activity of microparticles generated by collagen stimulation was significantly impaired in EDTA anticoagulated samples when compared with citrate. Microparticles were capable of higher degrees of thrombin generation than equivalent concentrations of phosphatidylserine (as assessed by XACT times), suggesting that other factors bound to the microparticle surface enhance the procoagulant response. In conclusion, the XACT assay is a specific method for the detection of procoagulant phospholipid activity arising from phosphatidylserine on the microparticle surface; however, other factors presumably bound to the surface of the microparticle may also contribute to enhanced thrombin generation detectable by prothrombinase assays.

Stored packed red blood cells contain a procoagulant phospholipid reducible by leukodepletion filters and washing

Background Ageing RBC gradually increase the exposure of phosphatidylserine (PS) on their surface, due to loss of membrane asymmetry. PS expression on red cells is not normally a significant factor in the hemostatic process, because aged RBC are rapidly cleared from the circulation. We propose that the presence of many altered red cells during massive transfusion can lead to increased procoagulant activity similar to what is seen in disease states where it is known to play a pathophysiologic role in microvascular disease. Study design and methods Procoagulant activity of phospholipid generated during storage of PRBC was evaluated using PRBC’s as the only source of phospholipid in the determination of modified Russell’s viper venom times of 10 PRBC units in which half of each unit was left unfiltered and half of each unit filtered. Florescent labeled annexin V binding by PRBC was also assessed by flow cytometry over time in storage. The effect of washing and filtration on these parameters was also determined. Results As time of storage increased, the Russell’s viper venom time of both the unfiltered and filtered units shortened ( p < 0.01). There was a significant lengthening of the Russell’s viper venom time at all time points measured when unfiltered units were compared to filtered units ( p < 0.01). In both unfiltered and filtered units, with increased length of storage, there was a gradual increase in the percentage of cells or particles binding annexin V ( p < 0.01). Filtration resulted in a significant reduction in the percentage of cells or particles binding annexin V at all time points measured ( p < 0.01). The effect of washing of PRBC units on the RVVT was assessed for unfiltered and filtered units on day 42. Washing resulted in a significant reduction of the RVVT in both unfiltered and filtered groups ( p < 0.01). Conclusions Levels of annexin V binding and procoagulant phospholipid activity similar to levels seen in disease states associated with significant vasoocclusive pathophysiology were found toward the end of the storage period of PRBC units. It was possible to reduce both of these parameters by leukodepletion at collection, and with washing of PRBC at the end of storage. Filtration at collection resulted in a 67% increase in RVVT over unfiltered units by day 42 of storage. On day 42 of storage, washing of filtered units resulted in a 21% increase in RVVT, and washing of unfiltered units resulted in a 34% increase in RVVT. The effects seen with filtration and washing were additive suggesting that in spite of filtration at collection, deterioration of cells continues based on age since further removal of phospholipid can be induced with washing of filtered units on day 42.

Increased procoagulant phospholipid activity in blood from patients with suspected acute coronary syndromes: a pilot study

Increased platelet activation is well documented in patients with acute coronary syndromes and can be detected by various methods, including flow cytometry and enzyme-linked immunosorbent assay. However, such techniques require several steps and cannot provide quick results. Platelet activation ultimately results in procoagulant phospholipid exposure and we have previously described a simple activated factor X-activated clotting time (XACT) test that is insensitive to resting platelets but that is significantly shortened by activated platelets, microparticles and procoagulant phospholipids. Our aim was to determine whether the XACT test could be used to distinguish patients with chest pain due to cardiac ischaemia from those having chest pain due to non-cardiac causes. We thus carried out XACT tests on ethylenediamine tetraacetic acid whole blood and plasma samples obtained from 46 patients presenting to the emergency department with chest pain and from 30 controls. Sixteen cases (30%) were subsequently diagnosed as acute coronary syndromes. Blood samples from these patients displayed overall significantly shortened XACT results relative to both healthy controls (P<0.001) and chest pain not due to cardiac ischaemia (P<0.004). This discrimination was much better with whole blood samples than when platelet-poor plasmas were tested (P=0.153), suggesting that free microparticles were not the only factors responsible. Thus, the detection of increased procoagulant phospholipid activity in whole blood by shortened XACT results may be a simple and rapid diagnostic marker of some cardiac ischaemic events.

The Influence of Fibrinogen and Fibrin on Thrombin Generation - Evidence for Feedback Activation of the Clotting System by Clot Bound Thrombin

In plasma the bulk of thrombin generation takes place after a clot has formed. We therefore investigated in what way the clot influences thrombin generation in plasma. The forming clot withdraws thrombin from free solution. Consequently less thrombin activity is found and less thrombin-inhibitor complexes are formed. The thrombin that is adsorbed to the clot reduces the lag time before thrombin generation in intrinsically or extrinsically triggered platelet poor plasma as well as in platelet rich plasma. We investigated the mechanism of this activation. Clots were obtained by recalcification of plasma or by the addition of thrombin-like enzymes (Reptilase, Agihal) from snake venoms. They were thoroughly washed until the washing fluid was devoid of any detectable clotting enzyme activity. In platelet poor plasma (PPP), thrombin-induced clots shorten the factor Va-dependent lag-time of thrombin generation in the extrinsic system as well as the factor VIIIa-dependent thrombin generation in the intrinsic system. Factor V or factor VII preparations that in itself hardly influence thrombin generation patterns acquire the capacity to shorten these lag-times when incubated with clot. The last washing fluid of the clot is inactive. Snake venom induced clots are not active either. Clots that are incubated in heparinised plasma for 1 h or more are as active as clots from normal plasma are. A role of factor Xa can not be excluded but must be minor because a clot made by addition of thrombin to plasma from which the factors II, VII, IX and X have been removed is as active as a clot from normal plasma is.(ABSTRACT TRUNCATED AT 250 WORDS)