Platelet Prothrombinase Activity Research Articles

Influence of Different Antithrombotic Regimens on Platelet-Mediated Thrombin Generation in Patients with Left Ventricular Assist Devices.

We characterized the biologic background of prothrombotic platelet function in the setting of durable left ventricular assist devices (LVADs) evaluating the role of different antithrombotic regimens. Platelet-mediated thrombin generation was quantified using the Platelet Activity State (PAS) Assay and the Thrombin Generation Test (TGT) in 78 patients implanted with the HeartMate II (n = 10, 13%), the HeartMate 3 (HM3) (n = 30, 38%), or the HVAD (n = 38, 49%) and managed with oral anticoagulation plus aspirin (n = 46, 59%) or anticoagulation alone (n = 32, 41%). Coagulation parameters (platelet count, International Normalized Ratio (INR), activated Partial Thromboplastin Time, Fibrinogen and D-Dimer levels) and hemolysis (lactate dehydrogenase levels [LDH]) were also recorded to comprehensively characterize the hemostatic profile in the two groups. In patients without aspirin, the PAS assay revealed low-intensity increase in platelet prothrombinase activity (1.11-fold, p = 0.03). Similarly the TGT revealed moderate higher platelet reactivity when compared with patients receiving aspirin, consistent with reduction in lag time (0.87-fold, p < 0.001), increase in peak of thrombin generation (1.5-fold, p = 0.002) and thrombin generation rate (2-fold, p = 0.02), but comparable endogenous thrombin potential (p = 0.50). Coagulation parameters and LDH were comparable in the two groups (p > 0.05). Moreover, no differences were noted in platelet prothrombinase activity of patients implanted with the HM3 or HVAD. Our results suggest that, in the setting of durable LVADs, aspirin minimally modulates the biochemical pathway of platelet-mediated thrombin generation. Accordingly, re-evaluation of current antithrombotic management criteria in patients stratified according to bleeding/thromboembolic risk might be safe and beneficial to prevent adverse events.

P2Y12 Receptor Antagonists: Do They Really Inhibit Shear-Mediated Platelet Activation within MCS?

Purpose Shear-mediated platelet activation (SMPA) by supra-physiological blood flow found within MCS is a main driver and contributor of device-related thrombosis. Thus, antiplatelet drugs targeting the main pathways of platelet activation are routinely prescribed. We have shown, that aspirin and GP IIbIIIa blockers have limited ability in mitigating SMPA, and do NOT protect platelets from integrin shedding and microparticle (MP) generation. In contrast, ticagrelor, a direct P2Y12 purinergic receptor antagonist, drastically inhibits platelet prothrombinase activity induced by high shear. The aim of the study was to define the role of the P2Y12 pathway in SMPA and evaluate the effect of P2Y12 antagonists on platelet procoagulant activity and MP generation induced by high shear stress. Methods The following P2Y12 antagonists were used: clopidogrel active metabolite (10 µM), cangrelor (1 µM) and ticagrelor (1 µM). After 10 min incubation with the agent, human platelets were exposed to continuous high shear stress (70 dyn/cm2, 10 min) in a hemodynamic shearing device. Platelet procoagulant activity was assessed using the annexin V binding assay, detecting procoagulant surface exposure, and platelet activity state (PAS) assay measuring thrombin generation. MPs were quantified by flow cytometry as CD41+/AnV+ particles distinguished from platelets by their FS/SS. Results Ticagrelor and cangrelor showed similar inhibitory effect, consistently decreasing both platelet procoagulant activity and microparticle generation induced by shear stress. The number of AnV+ platelets, AnV+ MPs, and PAS levels were decreased by 26%, 27%, and 30%, respectively, as compared with non-treated controls subjected to shear. Clopidogrel active metabolite, known for its low affinity to the targeted receptor, reduced PAS by 15%, while annexin V binding was diminished in more than 10 times for platelets and 2 times for MPs, indicating a significant reduction of platelet procoagulant surface exposure but limited inhibition of thrombin generation. Conclusion P2Y12 purinergic receptor antagonists inhibit shear-mediated platelet procoagulant surface formation and microparticle generation, both contributors in overall platelet prothrombotic activity induced by high shear stress within MCS. Our results suggest that P2Y12 pathway is involved in the regulation of SMPA.

Prothrombotic activity of cytokine-activated endothelial cells and shear-activated platelets in the setting of ventricular assist device support.

We systematically analyzed the synergistic effect of: (i) cytokine-mediated inflammatory activation of endothelial cells (ECs) with and (ii) shear-mediated platelet activation (SMPA) as a potential contributory mechanism to intraventricular thrombus formation in the setting of left ventricular assist device (LVAD) support. Intact and shear-activated human platelets were exposed to non-activated and cytokine-activated ECs. To modulate the level of LVAD-related shear activation, platelets were exposed to shear stress patterns of varying magnitude (30, 50, and 70 dynes/cm2, 10 minutes) via a hemodynamic shearing device. ECs were activated via exposure to inflammatory tumor necrosis factor-α (TNF-α 10 and 100 ng/ml, 24 hours), consistent with inflammatory activation recorded in patients on LVAD circulatory support. Adhesivity of shear-activated platelets to ECs was significantly higher than that of intact/unactivated platelets, regardless of the initial activation level (70 dynes/cm2 shear-activated platelets vs intact platelets: +80%, p < 0.001). Importantly, inflammatory activation of ECs amplified platelet prothrombinase activity progressively with increasing shear stress magnitude and TNF-α concentration: thrombin generation of 70 dynes/cm2 shear-activated platelets was 2.6-fold higher after exposure and adhesion to 100 ng/ml TNF-α‒activated ECs (p < 0.0001). We demonstrated synergistic effect of SMPA and cytokine-mediated EC inflammatory activation to enhance EC‒platelet adhesion and platelet prothrombotic function. These mechanisms may contribute to intraventricular thrombosis in the setting of mechanical circulatory support.

Procoagulant Platelet Subpopulation Formation in Whole Blood: Correlation with Whole Blood Clotting Time and 5-Year Mean Bleeding Score in Individuals with Factor VIII Deficiency.

Abstract 4022Poster Board III-958Different clinical phenotypes are often observed among individuals with hemorrhagic or thrombotic disorders despite the presence of the same mutation and/or factor levels suggesting that additional genetic and/or environmental factors influence clinical presentation. As thrombin plays a central role in hemostasis, factors that affect an individual's ability to generate thrombin may lead to an increased risk of hemorrhage or thrombosis. The goal of this study was to assess procoagulant platelet formation in individuals with hemophilia A using a rapid, whole blood flow cytometric assay of prothrombinase complex assembly on platelets described previously. In this assay, Ca2+-dependent factor Xa binding to activated platelets is used as a marker of thrombin generating potential as it has been shown previously to correlate with platelet prothrombinase activity in a washed platelet system. Procoagulant platelet subpopulation formation in 15 men with varying degrees of factor VIII-deficiency was evaluated and compared to two independent measures of hemostatic competence: a 5-year mean bleeding score and whole blood clot time. In these individuals, the % activated platelets binding factor Xa in whole blood varied from 2.35 – 9.0% (n = 30), which is consistent with what is observed in unaffected individuals (1.5 – 41.5%, n = 136). Bleeding was scored independently by two experienced hemophilia nurses and one hematologist at Centre Hospitalier Universitaire Sainte Justine based on each individual's bleeding history, including hemarthroses, soft tissue hematoma, and annual factor VIII usage, and averaged. The 5-year mean bleeding scores in these individuals ranged from 0 – 20.4. Linear regression analyses indicated that in this population the % activated platelets binding factor Xa correlated indirectly with the 5-year mean bleeding score, where individuals with lower bleeding scores (i.e. less bleeding episodes and/or factor VIII usage) generated larger procoagulant platelet subpopulations. The time to clot formation in a tissue factor-dependent, contact pathway-suppressed whole blood clotting assay described previously, was also determined. The clot time, which was determined visually, and ranged from ∼3 – 7 min, also correlated indirectly, though less well, with the % activated platelets binding factor Xa in whole blood (r = -0.23). Thus, consistent with what was observed with the bleeding score, those individuals who clotted more quickly (i.e. exhibited a greater degree of hemostatic competence) generated more proacoagulant platelets. Platelet procoagulant subpopulation formation was also compared to other hematological measures. An inverse correlation was observed between the % activated platelets binding factor Xa and mean platelet volume (r = -0.347), suggesting that in this population, individuals with smaller platelets (i.e. platelets with fewer prothrombinase binding sites) may generate a greater number of platelets capable of assembling prothrombinase. In contrast, no correlation was observed between whole blood platelet number and % activated platelets binding factor Xa. Interestingly, factor Xa binding was also negatively correlated with plasma levels of factor IX (r = -0.56) and factor V (r = -0.62). Platelet subpopulation formation was also weakly and negatively associated with the aPTT (r = - 0.28). In this small population of individuals with hemophilia A, whole blood platelet factor Xa binding correlates with bleeding phenotype. These observations support the notion that this measurement can be used to predict an individuals propensity to bleed or clot. Disclosures:No relevant conflicts of interest to declare.

Preservation of Platelets by Adding Epigallocatechin-3-O-Gallate to Platelet Concentrates

The effect of epigallocatechin-3-O-gallate (EGCG), a major component of green tea, on platelet preservation was evaluated. Single donor platelets (N = 10) were collected and preserved by the standard method. EGCG was added to the platelet concentrates before preservation and then the functional and biochemical parameters were monitored throughout the storage period. After 6 days of preservation, the aggregability of the platelets was significantly maintained by addition of 50 and 100 microg/ml of EGCG. Platelet prothrombinase activity was also significantly retained by the addition of EGCG. The accumulation of P-selectin and RANTES in the plasma preserved with EGCG was less than those preserved without EGCG, which indicated that EGCG might inhibit platelet activation. Furthermore, EGCG reduced the increase of LDH in plasma during preservation and inhibited the activation of caspase-3 and cleavage of gelsolin, thereby showing that EGCG could inhibit the apoptosis of platelets. These results suggest that EGCG may play an effective role in preserving platelets by inhibiting the activation and apoptosis of platelets.

Impaired Platelet Procoagulant Mechanisms in Patients with Bleeding Disorders

Activated platelets contribute to the arrest of bleeding by forming aggregates at sites of vascular injury and by providing a surface for assembling enzyme complexes involved in fibrin formation (platelet procoagulant activity; PCA). Impairment in the latter property of platelets has been observed in some disorders of hemostasis. In Scott syndrome, there is a defect in membrane vesiculation and in the surface expression of phosphatidylserine (PS), the phospholipid that is necessary for assembling the factor VIIIa/IXa (tenase) and factor Va/Xa (prothrombinase) complexes involved in thrombin formation. A family with an isolated defect in vesiculation, but normal prothrombinase activity, has also been reported. In the Quebec platelet disorder, overexpression of the fibrinolytic enzyme urokinase-type plasminogen activator results in the degradation of alpha-granule proteins, including factor V, and a specific abnormality in platelet factor V is the basis for the prothrombinase defect in platelet factor V-New York. The impaired prothrombinase activity in patients with delta-storage pool deficiency may be due to a failure to provide sufficient amounts of secreted adenine nucleotides which, when bound to P2 purinergic receptors, are necessary to maintain the intracellular Ca (2+) levels that are required for the surface expression of PS. Platelet prothrombinase activity and thrombin potential in patients with Glanzmann thrombasthenia (GPIIb-IIIa deficiency) may be decreased, normal, or increased, depending on the experimental conditions, for reasons that are not currently clear. The most consistent platelet PCA abnormality in the Bernard-Soulier syndrome (GPIb-complex deficiency) is an abnormally short serum prothrombin time, associated with a defect in the process by which an interaction between fibrin, von Willebrand factor, and GPIb promotes PCA.

Lactadherin blocks thrombosis and hemostasis in vivo: correlation with platelet phosphatidylserine exposure.

Platelet membrane phosphatidylserine (PS) is considered to be essential for hemostasis and thrombosis, but the in vivo topography of platelet PS has not been characterized. We hypothesized that platelet PS exposure would be identified on adherent platelets at the site of vascular injury and that blockade of PS would impede hemostasis and thrombosis. To localize and estimate the extent of platelet PS exposure and evaluate the impact of PS blockade in vivo. Lactadherin, a PS-binding milk protein, was utilized together with annexin V to detect both partial and complete membrane PS exposure on platelets in a mouse model of thrombosis and to evaluate the functional need for PS. Preliminary experiments were performed with synthetic membranes and with purified platelets. The number of lactadherin-binding sites on synthetic membranes was proportional to PS content, whereas annexin V required a threshold of 2.5-8% PS. Approximately 95% of thrombin-stimulated platelets exposed PS, but the quantity was below the threshold for annexin V binding at physiologic Ca(2+) concentrations. In mice, most adherent and aggregated platelets on the walls of ferric chloride-treated mesenteric veins exposed low levels of PS, rather than having complete exposure. In mice, blockade of PS with lactadherin inhibited platelet prothrombinase and factor Xase activity, and prolonged tail bleeding time and the time to carotid artery thrombosis. In vivo PS exposure contributes to both hemostasis and thrombosis. In this model of vascular injury, most platelets exhibit partial rather than complete PS exposure.

Critical Role for Cyclophilin D and the Mitochondrial Permeability Transition Pore (MPTP) in Platelet Activation.

Dual stimulation of platelets with thrombin and collagen results in the formation of a unique subpopulation of highly activated platelets. Characteristics of the highly activated platelet subpopulation includeincreased surface retention of procoagulant alpha granule proteins,high-level phosphatidylserine (PS) externalization, andmodulation of the fibrinogen receptor αIIbβ3 as evidenced by their decreased recognition by antibodies to activated αIIbβ3 such as PAC-1 and JON/A.Formation of the highly activated platelet subpopulation is closely correlated with a rapid loss of mitochondrial transmembrane potential (ΔΨm), a marker of MPTP formation. To test whether formation of the MPTP might regulate the development of the highly activated platelet subpopulation, platelet activation responses were examined in the presence of inhibitors and activators of MPTP formation. Cyclosporine, an inhibitor of MPTP formation, inhibited both PS externalization and αIIbβ3 modulation following dual stimulation with thrombin and the glycoprotein VI agonist convulxin (58 ± 4% vs. 9 ± 3%, p<0.01). Conversely, thrombin stimulation of platelets in the presence of H2O2 (100μM), an MPTP activator, increased PS externalization and αIIbβ3 modulation relative to platelets stimulated with thrombin alone (11 ± 3% vs. 48 ± 6%, p<0.05).Platelet activation responses were examined in cyclophilin D null (CypD −/−) mice, which have marked impairment of MPTP formation. Following dual agonist stimulation with thrombin and convulxin, both αIIbβ3 modulation and platelet PS externalization were significantly abrogated in CypD −/− platelets relative to wild type (7 ± 1% vs. 69 ± 1%, p<0.01). Alpha granule release, however, was unaffected in the absence of CypD. In vitro tests of platelet function similarly demonstrated that CypD −/− platelets had marked impairment of platelet prothrombinase activity relative to wild-type platelets after stimulation with thrombin and convulxin, but normal platelet aggregation responses.We then tested the hypothesis that CypD −/− mice would have an altered thrombotic response to arterial injury. Following photochemical injury of the carotid artery endothelium, a stable occlusive thrombus formed more rapidly in CypD −/− than in wild-type mice (16 ± 2 vs. 32 ± 7 min, p<0.05). Tail-bleeding time was unaffected. These results strongly implicate cyclophilin D and the MPTP as critical regulators of the subset of platelet activation responses occurring in the highly activated platelet subpopulation and suggest that activation of this novel platelet mitochondrial signaling pathway might play an important role in the regulation of the thrombotic response in vivo.

Platelet ABCA1 Expression Is Normal in Scott Syndrome Dogs.

Scott syndrome is a rare platelet function defect characterized by a failure of Ca2+-stimulated phosphatidylserine (PS) externalization and lack of platelet prothrombinase activity. In 2005 Albrecht et al. (Blood 106:542) described low levels of ATP-binding transporter A1 (ABCA1) mRNA in lymphocytes and heterozygosity for a novel ABCA1 missense mutation in a patient with Scott Syndrome. The proband's expression of platelet ABCA1, however, was not measured. Platelet dysfunction has been described as a feature of Tangier disease, a monogenic disorder caused by ABCA1 mutations (Nofer et al, JBC 279:34032, 2004). The disease phenotype results from altered lipid trafficking, with a virtual absence of plasma HDL cholesterol and accumulation of lipid in cells of the RE system. In the current study, we systematically evaluated platelets from Scott syndrome dogs to find evidence of ABCA1 deficiency. Platelets from control dogs and affected German shepherd dogs (GSD) were compared with regard to cholesterol content, ABCA1 protein expression, and reactivity to collagen, convulxin (CVX), and thrombin. All GSD had normal plasma cholesterol and triglyceride levels, with no significant difference in mean platelet cholesterol content from that of control dogs (control cholesterol = 13.4 ug/108 platelets, GSD cholesterol = 12.6 ug/108 platelets; n= 6; p = 0.726). Western blot analysis of platelet extracts from control dogs and GSD using a polyclonal antibody to human ABCA1 revealed comparable levels of a 220 kD protein band, the expected size for human ABCA1. We found no significant differences in maximal platelet aggregation in response to 12 ug/mL collagen stimulation [control dog mean response = 82.9% (n=10), GSD = 75.0 % (n= 7); p = 0.133)]. CVX concentration-response aggregation curves obtained for washed platelets were similar for control dogs (n=4) and GSD (n=3) in the range of 1 to 100 nM CVX, with a plateau of maximal aggregation at 10 nM CVX. In flow cytometry analyses, we found no difference in mean platelet mepacrine uptake between control dogs and GSD (mean positive platelets = 64%, n= 3 control, n=5 GSD). There were no significant differences in mean CD62P expression post-thrombin (0.5 U/mL) or CVX (2 nM) stimulation [control CD62P positive platelets post-thrombin = 57%, post-CVX = 47.5% (n=7); GSD CD62P positive platelets post-thrombin = 49%, post-CVX = 56% (n=4)]. Although ABCA1 has been proposed to play a role in PS transport, we found no evidence of ABCA1 deficiency and none of the characteristic platelet abnormalities associated with Tangier disease in canine Scott syndrome, a model of impaired transmembrane PS movement.Comparison of Platelet PhenotypesANALYSISTANGIER DISEASESCOTT SYNDROME GSDIonophore induced PS expressionPresentAbsentCollagen or CVX induced aggregationDecreasedNormalCVX or thrombin induced alpha granule releaseDecreasedNormalPlatelet ABCA1 expressionAbsentPresent

Convulxin Binds to Native, Human Glycoprotein Ibα

Convulxin (CVX), a C-type snake protein from Crotalus durissus terrificus venom, is the quintessential agonist for studies of the collagen receptor, glycoprotein VI (GPVI) and its role in platelet adhesion to collagens. In this study, CVX, purified from venom, behaves as expected, i.e. it binds to platelet GPVI and recombinant human GPVI, induces platelet aggregation and platelet prothrombinase activity, and binds uniquely to GPVI in ligand blots of SDS-denatured proteins. Nonetheless, we find that CVX has a dual specificity for both GPVI and native but not denatured human GPIb alpha. First, CVX binds to human GPIb alpha expressed on the surface of CHO cells. Second, CVX binds weakly to murine platelet GPIb alpha but more strongly to human platelet GPIb alpha, as evidenced by comparative binding to wild-type, GPVI(-/-), FcR gamma (-/-), and human GPIb transgenic mice. Third, the binding of CVX to human GPIb alpha is inhibited by soluble, recombinant human GPVI. Fourth, CVX binding to GPIb alpha is disrupted by phenylalanine substitutions at GPIb alpha tyrosine-276, tyrosine-278, and tyrosine-279, which also disrupts von Willebrand factor and alpha-thrombin binding to GPIb alpha. Fifth, CVX binding to GPIb alpha on Chinese hamster ovary cell transfectants is inhibited by function-blocking murine monoclonal anti-GPIb alpha antibodies. Lastly, CVX fails to bind to denatured GPIb alpha in detergent extracts of platelets. Three separate preparations of CVX (two purified by the authors; one obtained commercially) produced equivalent results. These results indicate that CVX exhibits dual specificity for both native GPIb alpha and GPVI. Furthermore, the binding site on GPIb alpha for CVX may be close to that for von Willebrand factor. Therefore, a contribution of GPIb alpha to CVX-induced platelet responses needs to be carefully re-evaluated.

Influence of platelet collagen receptor polymorphisms on risk for arterial thrombosis.

Collagens are major components of the vascular subendothelium, and the interaction of platelets with collagens initiates normal hemostasis or pathologic arteriothrombosis. Genetic factors that affect the interaction of platelets with collagens could represent risk factors for either arteriothrombosis or excessive hemorrhage. In this regard, we first found that platelet levels of one of the major platelet collagen receptors, integrin alpha(2)beta(1), vary up to 10-fold in normal healthy individuals and that the higher-level phenotype is associated with allele 1 (807T) of the integrin alpha(2) gene. More recently, we found that there is roughly a fivefold range in platelet glycoprotein VI content among normal individuals, which may also influence risk for thromboembolism. To determine if genetic polymorphisms of platelet glycoproteins involved in collagen-related function are associated with higher risk for thrombotic disorders, such as coronary heart disease, myocardial infarction, or stroke. We examined the genetic mechanisms responsible for variation in expression levels of the collagen receptor integrin alpha(2)beta(1) and the potential influence of this variation on risk for thrombotic diseases. We found that patients with arteriothrombotic diseases have a higher frequency of alpha(2) allele 1 (associated with higher levels of platelet integrin alpha(2)beta(1)). We further found that platelet glycoprotein VI content directly correlates with platelet prothrombinase activity, suggesting that a higher phenotype of platelet glycoprotein VI also may contribute to increased risk of arteriothrombotic diseases. Genetic polymorphisms that influence the level or function of platelet collagen receptors need to be seriously considered as genetic risk factors for arteriothrombotic diseases.

Effects of fibrinogen residence time and shear rate on the morphology and procoagulant activity of human platelets adherent to polymeric biomaterials.

Fibrinogen readily adsorbs to the surface of biomaterials and, because of its demonstrated ability to support platelet adhesion and aggregation, plays a role in thrombotic events associated with the implantation of synthetic materials in the human body. Thus, understanding the factors influencing the interactions of fibrinogen with biomaterials, and how platelet responses are affected, is crucial for the development of synthetic materials exhibiting improved blood compatibility. In this study, the effects of fibrinogen residence time and shear rate on the procoagulant activity of adherent platelets, along with their morphologic status, as deduced from scanning electron microscopy, were investigated. To examine whether adherent platelets promoted the generation of thrombin, polymeric materials (polytetrafluoroethylene, polyethylene, and silicone rubber) preadsorbed with fibrinogen were exposed to platelet suspensions at different wall shear rates and then incubated with clotting factors for 5 minutes under static conditions. The amount of thrombin generated per platelet was calculated from the optical density of the color developed by adding substrate S-2238. Scanning electron microscopy images of the platelets revealed that the platelets exhibited different morphologies, depending on the shear rate and residence time of the adsorbed fibrinogen. Platelets ranged from their normal discoid shape observed primarily under static conditions, to that of fully spread platelets. Results from this study show that platelets, in the presence of shear forces, undergo activation on exposure to surfaces on which adsorbed fibrinogen has resided for short residence times rather than long residence times. Interestingly, studies examining the procoagulant responses of such adherent platelets demonstrated that the platelets attached to the fibrinogen coated materials did not promote significant thrombin generation. Such low prothrombinase activity of adherent platelets suggests that adsorbed fibrinogen, while capable of supporting platelet adhesion and spreading on biomaterials, does not necessarily enhance the procoagulant activity of adherent platelets.

Modified Phosphatidylethanolamine as the Active Component of Oxidized Low Density Lipoprotein Promoting Platelet Prothrombinase Activity

We analyzed the influence of the atherogenic oxidized low density lipoproteins (LDL) on the activity of the platelet prothrombinase complex, a major contributor to overall thrombin formation in vivo. Platelet dependent thrombin generation was found to be strongly stimulated by in vitro oxidized LDL. The enhancement was additive to that observed with the platelet agonist thrombin. Oxidized LDL increased the platelet binding of annexin-V, suggesting that the augmented surface exposure of aminophospholipids promoted the prothrombinase activity. All of the stimulatory activity of the oxidized LDL could be recovered in the microemulsions prepared from the lipid portion of the modified particles. Phospholipid vesicles were prepared containing the total lipids of the oxidized LDL but lacking specifically in one lipid component. Following the selective removal of the ethanolamine phospholipids (PE) from the LDL lipids, the platelet-dependent thrombin formation was markedly reduced. Vesicles enriched with the isolated PE fraction alone enhanced the thrombin generation. Analyses with autoxidized phospholipids indicated that oxidation products of unsaturated diacyl-PE were mainly responsible for the increased prothrombinase activity. Oxidized LDL and its PE fraction lost their stimulatory activity after treatment with NaCNBH(3), a chemical reductant of Schiff base adducts. Phospholipid vesicles supplemented with synthetic aldehyde-PE adducts largely reproduced the stimulation of the thrombin generation. We conclude that the oxidized LDL particles elicit a pronounced prothrombotic response by increasing the activity of the platelet prothrombinase complex. Specific oxidative modifications of the LDL-associated ethanolamine phospholipids are mainly responsible for this stimulation.

The effect of storage on the expression of platelet membrane phosphatidylserine and the subsequent impacton the coagulant function of stored platelets.

Platelet concentrates (PCs) derived from whole blood and stored under standard blood bank conditions undergo changes that are referred to as the platelet storage lesion. This study assesses the effect of PC preparation and storage on the distribution of phosphatidylserine (PS) in the platelet membrane and the effect that this distribution may have on the thrombogenic potential of stored PCs. Fresh platelets and PCs donated by healthy donors were obtained. PCs derived from platelet-rich plasma were studied on Day 1, Day 3, and Day 6 of storage under blood bank conditions. Platelet aggregation after exposure to the platelet agonists ADP and epinephrine singly declined progressively, but, when ADP and epinephrine in combination and collagen and thrombin in combination were used as agonists, the decline in platelet aggregation was less marked. PS expression as measured by Annexin V binding (mean and SD) was 2.02 +/- 0.93 percent in fresh platelet samples and increased to 5.39 +/- 4.2 percent on Day 1, 22. 1 +/- 7.1 percent on Day 3, and 39.5 +/- 12.1 percent on Day 6. Platelet prothrombinase activity (mean +/- SD) as measured by thrombin generation increased from 1.49 +/- 0.7 micro per mL in fresh platelet samples to 3.68 +/- 1.1 micro per mL in Day 1 platelets (p<0.001), 5.15 +/- 2.5 micro per mL in Day 3 platelets (p<0.001), and 4.65 +/- 2.48 micro per mL in Day 6 platelets (p<0. 001). These results show that PS expression increases after preparation of PCs from platelet-rich plasma and rises progressively during platelet storage under blood bank conditions. Furthermore, the greater PS expression is associated with increased platelet- dependent thrombin-generating capacity.

Platelet prothrombinase activity, a final pathway platelet procoagulant activity, is overexpressed in type 1 diabetes: no relationship with mean platelet volume or background retinopathy.

There is overwhelming evidence that platelets from diabetic individuals are hyperreactive, not only when microvascular complications are apparent, but already at an early stage of the disease. There is still controversy about the question of whether primary hyperreactive platelets may contribute to the origin or progression of microangiopathy or whether diabetic platelet hyperfunctionality is just a logical consequence of a continuous low-grade activation of platelets by contact with a diseased microvascular wall. As a consequence of platelet activation, the outer layer of its phospholipid membrane is more procoagulant than in the quiescent state, stimulating thrombin formation in plasma. This platelet function is called platelet procoagulant activity. We studied platelet prothrombinase activity (PPA), a final pathway platelet procoagulant activity of type 1 diabetic platelets, and looked for an eventual correlation with microvascular disease (background retinopathy) and mean platelet volume (MPV). Stypven clotting times (SCTs), reflecting PPA expression, and MPV of citrated platelet-rich plasma (PRP), were measured in 21 patients with type 1 diabetes-10 with and 11 without background retinopathy-under clinically acceptable metabolic control and compared them to 20 disease-free voluntary controls. We also compared PPA expression and MPV in diabetic individuals with and without retinopathy. With the SCT, a selective test adapted for studying PPA in PRP, we found hyperexpression of PPA in all diabetic patients. We found no difference in MPV between diabetic and control PRP. Comparing patients with and without background retinopathy we found no significant difference in PPA expression. From these results, we suggest that the phospholipid surface of diabetic platelets, more than the surface of normal control platelets, stimulate the expression of PPA. This diabetic platelet coagulant anomaly was not related to an increased platelet mass (higher MPV) nor to the presence of microangiopathy. We conclude that PPA hyperexpression is associated with patients with type 1 diabetes, already occurring in an early stage of the disease, and not necessarily a consequence of early-stage microvascular disease, because the anomaly is also demonstrable, in the same degree, in patients with diabetes without microangiopathy.

Enoxaparin, a low molecular weight heparin, inhibits platelet-dependent prothrombinase assembly and activity by factor-Xa neutralization.

The available evidence suggests strongly that intravascular thrombosis is mediated predominantly by tissue-factor and its activation of factor X, which in the presence of factor Va, calcium, and phospholipid (prothrombinase complex) effectively converts prothrombin to thrombin. In vitro experiments have shown that low molecular weight heparins (LMWHs) have greater anti-Xa activity than unfractionated heparin; however, it remains unclear as to whether their antithrombotic effects in vivo are determined by a similar mechanism. We determined the ability of plasma obtained from patients with either unstable angina or non-ST segment elevation myocardial infarction (MI) receiving the LMWH enoxaparin (anti Xa:IIa ratio 3:1) to inhibit tissue factor-mediated thrombin generation and to inactivate platelet prothrombinase. Platelet rich plasma was prepared by suspending washed donor platelets in the plasma of 7 patients participating in the TIMI 11A study. Samples were obtained before, 1 hour after a 30-mg IV bolus of enoxaparin and 6 hours after the third subcutaneous injection (1. 0-1.25 mg/kg given subcutaneously every 12 hrs). Tissue factor (0.1 ng/ml) and 10 mM CaCl(2) were added to initiate extrinsic coagulation. At timed intervals prothrombin activation fragment 1.2 (F1.2) levels (thrombin generation) were measured using an ELISA technique. Inactivation of reformed platelet prothrombinase by samples obtained at the same time points was also determined. Patient plasma obtained 1 hr after treatment initiation and 6 hours after the third subcutaneous injection inhibited tissue factor mediated prothrombinase assembly by 31% and 11%, respectively and platelet prothrombinase activity by 27% and 22%, respectively. We conclude that enoxaparin in plasma concentrations achieved routinely in clinical practice is able to: (1) inhibit tissue factor mediated extrinsic coagulation by preventing platelet surface prothrombinase assembly, and (2) inactivate platelet prothrombinase activity and resulting thrombin generation. These observations suggest that a LMWH's anti-Xa activity (and anti-Xa:IIa profile) is important in determining its overall antithrombotic potential. Clinical trials comparing agents with differing anti-Xa:IIa properties will be required, however, to provide proof of concept.

Platelet Agonists Enhance the Import of Phosphatidylethanolamine into Human Platelets

It is unknown whether the endocytosis-independent transfer of phospholipids from lipoproteins to platelets is regulated by platelet agonists such as thrombin. The movements of the choline phospholipids phosphatidylcholine and sphingomyelin (labeled with either 14C or the fluorescent pyrenedecanoic acid) between low density lipoproteins and platelets were unaffected by thrombin (0.5 unit/ml). In contrast, thrombin accelerated the import of diacyl phosphatidylethanolamine (PE) and alkenylacyl phosphatidylethanolamine into platelets by about 4-fold. Similarly, thrombin receptor-activating peptide (15 microM), collagen (10 microgram/ml), and ADP (10 microM) enhanced PE uptake. High density lipoprotein particles and egg phosphatidylcholine vesicles were also donors for stimulation of platelet PE import. Part of the [14C]arachidonic acid-labeled PE transferred from low density lipoprotein to platelets activated by thrombin and collagen was metabolized to 14C-eicosanoids. Inhibitors of protein kinase C partially prevented thrombin-induced [14C]PE uptake, while direct activators of protein kinase C increased incorporation of [14C]PE into platelets. Proteinaceous factor(s) recovered in the extracellular medium from ADP- and thrombin-activated platelet suspensions were found to accelerate the transfer of pyrenedecanoic acid-labeled PE between donor and acceptor lipid vesicles. The stimulation of import of ethanolamine phospholipids led to a 2-fold enhancement of the prothrombinase activity of thrombin-activated platelets. Our study demonstrates that physiological platelet stimuli increase specifically the transfer of ethanolamine phospholipids from lipoproteins to platelets through a secretion-dependent mechanism. This might contribute to the increase of procoagulant activity of stimulated platelets.

Glycoprotein IIb/IIIa Receptor Antagonists Inhibit the Development of Platelet Procoagulant Activity

We examined the effects of glycoprotein IIb/IIIa (GPIIb/IIIa) antagonists c7E3 Fab and DMP728 on the development of platelet prothrombinase (PT) activity. c7E3 Fab dose-dependently inhibited the rate of thrombin-stimulated thrombin generation over a 1-minute reaction time. The IC50 was 11 nM with an IC90 of 1000 nM. DMP728 inhibited PT activity maximally by 60% at 100 nM. A similar profile was observed for the inhibition of platelet tenase activity. Inhibition was platelet specific up to approximately 200 nM c7E3 Fab. Above 200 nM, inhibition was platelet independent, as shown by the inhibition of activity assembled on PS/PC vesicles. c7E3 Fab and DMP728 did not inhibit calcium ionophore-induced activity. DMP728 potency diminished with reaction time (over 6 minutes) whereas c7E3 Fab potency did not. Inhibition by 2 μM DMP728 was not further increased by 20 nM c7E3 Fab. Heparin inhibition of platelet PT activity was additive to that of c7E3 Fab. Studies with added von Willebrand factor (vWf) indicate that in the context of thrombin activation vWf activates platelets through mechanisms independent of GPIIb/IIIa to promote PT activity. Thrombin activation induced binding of FITC-AnnexinV to a subpopulation of platelets which was reduced by approximately 50% by pretreatment with either c7E3 Fab or DMP728. Together, these data indicate that c7E3 Fab and DMP728 inhibit the development of GPIIb/IIIa-mediated platelet PT activity at events during platelet activation. The inhibitory activities are not additive, suggesting these agents compete for the same site or inhibit via the same mechanism. Inhibition accompanies a reduction in the number of phosphatidylserine binding sites, implying that GPIIb/IIIa receptor antagonists reduce platelet membrane scrambling induced by thrombin. The additivity of inhibition with heparin by c7E3 Fab suggests a combination of these agents might have a greater bleeding liability than the use of either agent alone.

Clinical Syndromes Associated with Lupus Anticoagulants

Recent evidence suggests that lupus anticoagulants are immunologically distinct from the anticardiolipin antibodies. Nevertheless, the associated clinical complications exhibited by the two groups of antibodies are similar. They have been shown to have a strong association with a history of arterial and venous thrombosis, thrombocytopenia and neurological disease in patients with SLE or lupus-like disorders. The association between antiphospholipid antibodies and recurrent fetal loss is suggested by the currently available data but is not firmly established. Patients with lupus and antiphospholipid antibodies and an established history of recurrent fetal wastage are at high risk for experiencing subsequent fetal loss, but it is not yet known whether the same is true for patients without a history of fetal loss. The association of thrombosis, neurological disease, thrombocytopenia, and fetal loss in patients with non-SLE disorders has not been as extensively studied. Only recently have investigators such as Ginsberg and colleagues begun to show in prospective studies that there may, in fact, be a statistically significant risk of thrombotic events in otherwise healthy individuals with antiphospholipid antibodies. Many of the diverse minor manifestations reported in individual patients, case series, or cross-sectional studies such as livedo reticularis, leg ulcers, and hemolytic anemia may, alternatively, be due to coincidence or chance. Efforts to elucidate the mechanisms of thrombosis in patients with antiphospholipid antibodies is an area of active research. Most efforts have been based on the effects of these antibodies on endothelial cell and platelet function as well as on the fibrinolytic system. In addition, it has recently been shown that binding of antiphospholipid antibodies to phospholipids requires the serum "co-factor" beta 2-glycoprotein I. In patients with SLE selected for the presence of the lupus anticoagulant, thrombosis, or fetal loss, Viard and associates found that 17 of 47 (36%) patients had anti-beta 2-glycoprotein I antibodies. They were able to show, in their small retrospective study, that there was an association between the presence of these antibodies and anticardiolipin activity, lupus anticoagulant activity, and thrombotic events, but not with spontaneous abortion. Of patients with SLE and thrombosis (9 of 47) eight of nine were positive for anti-beta 2-glycoprotein I antibodies, seven of nine were positive for anticardiolipin antibodies, and eight of nine were positive for the lupus anticoagulant. The known inhibitory effect of beta 2-glycoprotein I on platelet aggregation, on platelet prothrombinase activity, and on the intrinsic pathway of coagulation supports the hypothesis that implicates beta 2-glycoprotein I in the pathogenesis of unwanted thrombotic events.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of antiphospholipid antibodies on procoagulant activity of activated platelets and platelet‐derived microvesicles

We have recently described the in vitro mechanism of action of anticardiolipin (aCL) and lupus anticoagulant (LA) antibodies in patients with the antiphospholipid syndrome. LA antibodies inhibit coagulation reactions in plasma because they appear to recognize the complex of lipid-bound (human) prothrombin, whereas aCL antibodies require beta 2-glycoprotein I (beta 2-GPI) for binding to anionic phospholipids. aCL antibodies can be divided into two subgroups, according to their behaviour in lipid-dependent coagulation reactions: aCL-type A enhances the anti-coagulant effect of beta 2-GPI, whereas aCL-type B does not. In the present study we investigated the effect of purified aCL-type A and B and of LA antibodies on the procoagulant activity of both Ca-ionophore activated platelets and platelet-derived microvesicles, using an assay system with highly purified bovine coagulation factors Xa, Va, and prothrombin from human and bovine origin. In the absence of beta 2-GPI neither type of aCL was able to inhibit the prothrombinase activity of platelets or microvesicles. However, a strong and dose-dependent inhibition of the prothrombinase activity of both platelets and platelet-derived microvesicles was observed within a few minutes, when aCL-type A antibodies were added in combination with beta 2-GPI. This inhibitory effect was dependent also on the concentration of beta 2-GPI. Conversely, no inhibitory effect of aCL-type B antibodies on platelet- (or microvesicle) prothrombinase activity in the presence of beta 2-GPI could be observed. LA antibodies were able to inhibit in a dose-dependent way the procoagulant activity of activated platelets and platelet-derived microvesicles. With two LA preparations this inhibition was only apparent when human prothrombin was used as substrate, while a third preparation exhibited its inhibitory effect both in the presence of human and bovine prothrombin. The data indicate that, in the presence of their respective cofactors beta 2-GPI and prothrombin, aCL and LA antibodies interact with the membrane of activated platelets and platelet-derived microvesicles in a very similar way as previously observed for their interaction with anionic phospholipid surfaces.