Perioperative dilutional coagulopathy treated with fresh frozen plasma and fibrinogen concentrate: a prospective randomized intervention trial.

BACKGROUND AND OBJECTIVES: Treatment of dilutional coagulopathy by transfusing fresh frozen plasma (FFP) remains sub-optimal. We hypothesized that partial replacement of transfused FFP by fibrinogen concentrate results in improved coagulant activity and haemostasis. This was tested in a controlled clinical intervention trial with patients experiencing massive bleeding during major surgery. METHODS: Patients undergoing major elective surgery were treated according to current protocols. When transfusion with FFP was required, patients were randomized as follows: group A received 4 units FFP and group B received 2 units FFP plus 2 g fibrinogen concentrate. Blood samples were taken before and after the intervention. Analysts were blinded to the treatment type. RESULTS: Group A (B) consisted of 21 (22) patients, in 16 (17) of whom bleeding stopped after intervention. Plasma fibrinogen increased significantly more in group B (0·57 g/l) than in group A (0·05 g/l). However, levels of prothrombin and factors VIII, IX and X increased more in group A than in group B. Rotational thromboelastometry (ROTEM) of whole blood and plasma revealed improved fibrin clot formation in group B but not in group A. Thrombin generation [calibrated automated thrombogram (CAT)] in plasma increased more in group A. Principal parameters determining whole-blood thromboelastometry were the fibrinogen level and platelet count. In vitro addition of fibrinogen and prothrombin complex concentrate to pre-intervention samples restored both ROTEM and CAT parameters. CONCLUSIONS: Partial replacement of transfused FFP by fibrinogen increases fibrin clot formation at the expense of less improved thrombin generation. Coagulation factors other than fibrinogen alone are required for full restoration of haemostasis.

Acquired platelet antagonism: off-target antiplatelet effects of malignancy treatment with tyrosine kinase inhibitors.

Platelets can contribute to tumor progression and metastasis. Cancer patients are at increased risk of thrombosis, and advanced stages of cancer are associated with thrombocytosis or increased platelet reactivity. Tyrosine kinase inhibitors (TKIs) are widely used as a targeted strategy for cancer treatment, with the aim of prolonging progression-free survival of the patients. Because of their broad kinase target spectrum, most TKIs inevitably have off-target effects. Platelets rely on tyrosine kinase activity for their activation. Frequently observed side effects are lowering of platelet count and inhibition of platelet functions, whether or not accompanied by an increased bleeding risk. In this review, we aim to give insights into: (i) 38 TKIs that are currently used for the treatment of different types of cancer, either on the market or in clinical trials; (ii) how distinct TKIs can inhibit activation mechanisms in platelets; and (iii) the clinical consequences of the antiplatelet effects of TKI treatment. For several TKIs, the knowledge on affinity for their targets does not align with the published effects on platelets and reported bleeding events. This review should raise awareness of the potential antiplatelet effects of several TKIs, which will be enhanced in the presence of antithrombotic drugs.

PO-19 - Platelet (PLT) adhesion under flow condition in essential thrombocythemia (ET) and polycythemia vera (PV) is variably influenced according to patient mutational status.

INTRODUCTION: The myeloproliferative neoplasms ET and PV are characterized by a high incidence of both arterial and venous thrombosis, and/or microcirculatory disturbances. Three somatic mutations, i.e. JAK2-V617F, Calreticulin (CalR) and MPL, commonly found in these diseases, correlate with different thrombotic risk levels. AIM: To analyze the influence of JAK2-V617F, CalR and MPL mutations on PLT adhesion, evaluated by a dynamic method under flow conditions in a group of patients with ET and PV. MATERIALS AND METHODS: 86 patients, i.e. 51 ET (19 M/32 F; age range 32-86 years) and 35PV (22 M/13 F; 41-83 yrs.), and 24 healthy controls (13 M/11 F; 28-61 yrs.) were enrolled upon informed consent. For the adhesion assay, peripheral venous whole blood was perfused over collagen for 4' at a 1,000 s-1 shear rate. PLTs were then stained with an anti-P-selectin-FITC antibody to evaluate PLT activation, and annexin V-AlexaFluor647 to detect procoagulant phosphatidylserine expression. Then, images of adherent PLTs in random fields were taken using phase contrast and fluorescence imaging by EVOS® fluorescence microscope. Results are mean±SEM of the % area covered by PLTs, or as the % of adherent PLTs positive for P-selectin or phosphatidylserine. Main hematological parameters and mutational status were recorded. RESULTS: PLT adhesion was significantly (p<0.01) greater in ET (44.6±1.6%) and PV patients (49.0±1.9%) compared to controls (37.9±1.7%). In ET, PLT adhesion was highest in JAK2-V617F mutation carriers (n=23), followed by CalR-positive (n=16) and triple negative subjects (n=9), and lowest in the MPL-positive patients (n=3). In PV, no difference in PLT adhesion was observed between JAK2-V617F heterozygous and homozygous subjects. P-selectin expression by adherent PLTs was not statistically different between patients and controls. Differently, phosphatidylserine expression on adherent PLTs was significantly reduced (p<0.01) in both ET and PV compared to healthy subjects. In ET patients, a significant (p<0.05) correlation was found between PLT adhesion and PLT count in JAK2-V617F and CalR-positive mutation carriers. Multivariate regression analysis adjusted for age and sex, confirmed PLT count as a significant determinant of PLT adhesion in JAK2-V617F positive patients only. CONCLUSIONS: ET and PV platelets show an increased adhesion to collagen in vitro, particularly in those carrying the JAK2-V617F mutation. A prospective study is ongoing to evaluate the predictive value of our PLT thrombus formation dynamic model for the thrombotic risk in ET and PV patients. ACKNOWLEDGEMENT: Project funded by "AIRC-IG2013" grant Nr. 14505 from the "Italian Association for Cancer Research" (A.I.R.C.).

OC-08 - Multiple functional defects in platelets from thrombocytopenic cancer patients undergoing chemotherapy.

INTRODUCTION: Severe thrombocytopenia (≤50×10(9) platelets/L) is often the consequence of hematological malignancies and intensive chemotherapy. The risk of clinically significant bleeding is increased in these patients, despite the use of prophylactic platelet transfusions. The fact that there is no clear correlation between the platelet count and the risk of hemorrhage, suggests that there are other contributing factors. The contribution of impairments in platelet and coagulant function remains poorly understood. AIM: In patients with chemotherapy-induced thrombocytopenia due to hematological malignancies, we evaluate platelet and coagulant functions and determine the effects of platelet transfusion. Ultimately, we can identify specific hemostatic factors that aid in the prediction of bleeding. MATERIALS AND METHODS: In total 58 patients were included and blood was collected before and, if indicated (≤10×10(9) platelets/L), 1 hour after transfusion with platelet concentrate. Platelet function was assessed using flow cytometry by determining: 1) integrin αIIbß3 activation (PAC-1 antibody), 2) P-selectin expression (anti-P-selectin antibody), 3) phosphatidylserine exposure (Annexin-V) and 4) intracellular calcium (Fluo-4 AM). Factor levels were determined in plasma. Thrombus and fibrin formation was assessed by perfusion of whole blood over a collagen-tissue factor surface at a shear rate of 1,000 s-1. RESULTS: Platelets from the thrombocytopenic patients before transfusion showed markedly reduced integrin αIIbß3 activation and P-selectin expression in response to thrombin, collagen-related peptide and ADP, compared to healthy donor platelets. Also, agonist-induced intracellular calcium fluxes were greatly reduced. However, calcium fluxes with thapsigargin, a SERCA pump inhibitor, were similar in patient and control platelets, suggesting a normal calcium store content in the patient platelets. Furthermore, phosphatidylserine exposure was increased in unstimulated patient platelets compared to control platelets (8.2 vs. 1.8%, p<0.0001). Coagulation factor levels were within the normal range, with the exception of von Willebrand factor and fibrinogen levels, which were elevated. Platelet transfusion partly recovered the platelet integrin αIIbß3 activation and P-selectin expression induced by agonists. Platelet deposition (6.7 vs. 1.7%, p<0.0001) and fibrin formation (7.6 vs. 0.9%, p=0.0005) under flow conditions were substantially improved after platelet transfusion. CONCLUSIONS: Platelets from cancer patients undergoing chemotherapy appear to display impaired functional responses to activating stimuli. Platelet transfusion partly restores these functional defects, resulting in improved thrombus and fibrin formation.

Membrane fluidity of non-activated and activated human blood platelets.

The steady-state fluorescence anisotropy of membranes labeled with 1,6-diphenyl-1,3,5-hexatriene (DPH) or its 4'-trimethylammonio derivative, TMA-DPH, is generally considered a measure for the lipid order and, hence, inversely related to membrane fluidity. We now report that anisotropy values of DPH- and TMA-DPH-labeled human platelets are considerably influenced by experimental conditions like the platelet concentration, which do not affect membrane fluidity. Activation of platelets with thrombin increases, but activation with ionomycin decreases anisotropy values with both labels. Such anisotropy changes are not detected in platelet membranes or platelet lipids, when isolated after activation of the intact platelets. We present evidence that the anisotropy changes of intact platelets are not a consequence of modified lipid composition (e.g., as would be induced by phospholipase A2 activity) but are, at least partially, caused by changed optical properties of the cell suspension. Measurement of membrane fluidity of platelets by fluorescence polarization is severely hindered by a high turbidity of the platelet suspension and also by changes in the turbidity and platelet morphology during the activation process.

Effects of dietary fatty acids on signal transduction and membrane cholesterol content in rat platelets.

Previously, we have reported that dietary fatty acids can modify the thromboxane A2-dependent activation of rat platelets. Here, we present evidence that this dietary effect is part of a more general effect on platelet signal transduction, putatively involving structural changes in the platelet membranes. Four experiments were performed, where Wistar rats were fed with a high-fat diet enriched in either saturated, n-6 polyunsaturated or n-3 polyunsaturated fatty acids, or with a low-fat diet enriched in n-6 polyunsaturated fatty acids. The type of diet hardly influenced mean number of double bonds in the major platelet phospholipids. Platelet membranes from the rats fed with the saturated-fat diet had phospholipids with relatively high levels of arachidonate, but were low in cholesterol/phospholipid ratio. When compared to this diet group, platelets from other groups had an arachidonate content that was 21 to 47% lower and a cholesterol/phospholipid ratio 3 to 5% higher. The saturated-fat diet resulted in platelets that, in general, were less responsive to agonists than the platelets from other groups: with thrombin, collagen and thromboxane A2 analogue U46619, both early (shape change and phospholipase C-dependent rise in [Ca2+]i) and late (exocytosis and aggregation) responses were relatively low. However, platelet activation evoked by ADP was not influenced by diet type. When the cholesterol content of rat platelets was modified in vitro, it appeared that the early and late responses to thrombin and U46619 increased with the cholesterol/phospholipid ratio. Taken together, these results suggest that in rat platelets (i) the membrane cholesterol/phospholipid ratio can be modulated by a diet rich in saturated fatty acids, explaining, at least in part, the dietary effect on phospholipase C-mediated platelet activation, and (ii) relatively small changes in cholesterol content can have a more profound effect on platelet activation than substantial changes in arachidonate level.

Function of intracellular [Ca2+]i in exocytosis and transbilayer movement in human platelets surface-labeled with the fluorescent probe 1-(4-trimethylammonio)phenyl)-6-phenyl-1,3,5-hexatriene.

Ellipsometry indicated that 1-(4-(trimethylammonio)phenyl-6-phenylhexa-1,3,5-triene (TMA-DPH) bound to platelets in a reversible and saturable way. Accordingly, the fluorescence intensity (F) of a suspension of TMA-DPH-labeled platelets was described as a quantity, determined by the amount of TMA-DPH bound to the platelet surface. Most platelet activators elevated F to a degree that correlate well with the secretion of serotonin evoked by these activators. The increase in F levels reflected the increase in outer membrane surface area following exocytosis. However, activators that evoked prolonged (> 2.5 min) and strong (> 600 nM) elevations of cytosolic [Ca2+]i increased F to levels that were much higher than expected from the maximal increase in surface area due to exocytosis. This high increase in F was caused by inward transbilayer movement of TMA-DPH over the plasma membrane and the subsequent labeling of cytosolic membrane sides. The kinetics of exocytosis and changes in cytosolic [Ca2+]i were studied by stopped-flow mixing of platelets with agonist. Thrombin-induced exocytosis had a delay of only 3 s, which was shortened when external CaCl2 or ADP was present. This correlated well with a faster rise in [Ca2+]i in the presence of CaCl2 or ADP, indicating that exocytosis was linked in time to elevation of [Ca2+]i. By itself, ADP was unable to evoke exocytosis and it elicited a [Ca2+]i transient of much shorter duration than thrombin, but with similar maximum. We concluded that both exocytosis and transbilayer movement were associated with elevation of [Ca2+]i: exocytosis required a moderate, relatively prolonged rise and transbilayer movement was accompanied by a stronger rise of even longer duration. Influx of external Ca2+ was essential for transbilayer movement, but not for exocytosis.

Influence of dietary fatty acids on membrane fluidity and activation of rat platelets.

The apparent steady-state fluorescence anisotropy of DPH- or TMA-DPH-labeled washed rat platelets is strongly affected by factors that also influence the turbidity by these platelet suspensions. Sonicated preparations from platelet lipids have a low turbidity and give anisotropy values which are hardly affected by the experimental conditions. We studied the effect of four high-fat diets on membrane fluidity, lipid composition and activation tendency of washed platelets. The diets contained 50 energy% of oils with different levels of saturated and (poly)unsaturated fatty acids. Only small diet-induced differences in DPH fluorescence anisotropy were found, which were comparable for intact platelets and platelet lipids. These differences were unrelated to the degree of saturation of the dietary fatty acids. Platelets from rats fed mainly saturated fatty acids differed significantly from other diet groups in a higher unsaturation degree of phospholipids and a lower cholesterol/phospholipid ratio, but this was not detected by DPH in terms of decreased anisotropy. These platelets aggregated less than other platelets in response to thrombin or collagen. The lower response to collagen persisted in indomethacin-treated platelets activated with the thromboxane A2 mimetic U46619, indicating a different sensitivity of these platelets for thromboxane A2. We conclude that in rat platelets: (a) the overall membrane fluidity and phospholipid unsaturation degree are subject to strong homeostatic control; (b) steady-state anisotropy with DPH or TMA-DPH label is inadequate to reveal subtile changes in lipid profile; (c) changes in platelet responsiveness to thrombin and thromboxane A2, rather than (plasma) membrane fluidity, determine the effect of dietary fatty acids on platelet aggregation.

Effects of U73122 and U73343 on human platelet calcium signalling and protein tyrosine phosphorylation.

We have investigated the actions of the PLC inhibitor, U73122, and its close analogue, U73343, which does not inhibit PLC, in Fura-2-loaded human platelets. Rises in [Ca2+]i evoked by thrombin and collagen, and the TxA2-dependent rise in [Ca2+]i evoked by thapsigargin, were abolished by U73122, indicating that it inhibits the activity of both beta and gamma isoforms of PLC. The supposed control compound U73343, was found to inhibit TxA2 formation; it therefore partially inhibited the rise in [Ca2+]i evoked by low concentrations of thrombin, by thapsigargin or by collagen. U73343 had a greater effect than aspirin on the action of collagen, indicating an action on the TxA2-independent component of the signal, via PLC gamma-U73343 lowered TxA2 production by inhibiting the activation of cPLA2, probably at a tyrosine phosphorylation step. U73343 seems to inhibit only the tyrosine kinases involved in the activation of PLC gamma and the generation of TxA2. In contrast, U73122 increased tyrosine phosphorylation of platelet proteins, perhaps by inhibiting receptor independent tyrosine phosphatases, but inhibited all further tyrosine phosphorylation on addition of thrombin or other agonists.

Heterogeneity in microparticle formation and exposure of anionic phospholipids at the plasma membrane of single adherent platelets.

Adherent platelets were examined for their ability to form microvesicles and procoagulant sites for thrombin formation. Epifluorescence and phase-contrast microscopy were employed to visualize shape changes, changes in intracellular Ca(2+) levels ([Ca(2+)](i)), vesiculation of the plasma membrane and appearance of anionic phospholipids in the outer leaflet of the plasma membrane, as probed by annexin V binding. In the absence of extracellular Ca(2+) two stable populations of adherent platelets were observed. The majority of the adherent platelets were fully spread and about 10% remained in a non-spread dendritic state. In the presence of extracellular Ca(2+) vesiculation at the surface of spread platelets occurred at a rather slow rate (10% of the platelets after 20 min) concomitantly with an increase in [Ca(2+)](i) and binding of annexin V. However, a small fraction of the adherent platelets ( approximately 1%) responded much faster. Ionomycin-enhanced influx of Ca(2+) in dendritic platelets resulted in a rapid transformation of these platelets into inflated, balloon-shaped, platelets having a diameter of 2.0+/-0.7 microm without notable microvesicle formation. In contrast, fully spread platelets retained their shape but obtained frayed edges as a result of microvesicle formation. Confocal scanning fluorescence microscopy indicated that annexin V bound to very distinct sites at the outer plasma membrane of spread as well as balloon-shaped platelets. Inhibition of platelet calpain activity suppressed ionomycin-enhanced microvesicle formation and ballooning of platelets, but not annexin V binding. These findings indicate that vesiculation and ballooning, but not the exposure of phosphatidylserine at the outer leaflet of the adherent platelet membrane, are associated with cytoskeleton destruction. Altogether, the data suggest a similar relationship between [Ca(2+)](i) and the formation of platelet procoagulant sites as reported for platelets in suspension. However, the present investigations on single adherent platelets reveal for the first time that adhesion and spreading of platelets is not necessarily associated with the appearance of procoagulant sites. Secondly, an unexpected diversity was observed among adherent platelets with respect to sensitivity to Ca(2+)-induced generation of procoagulant sites and Ca(2+)-induced vesiculation of plasma membrane. It is tempting to speculate that this diversity is of importance for the procoagulant response of platelets to a hemostatic challenge elicited by an injured vessel wall.

Unstimulated platelets evoke calcium responses in human umbilical vein endothelial cells.

Interactions between human platelets and human umbilical vein endothelial cells (HUVEC) were studied by monitoring changes in cytosolic [Ca2+]i in both cell types. Confluent monolayers of Fura-2-loaded HUVEC, grown on gelatin-coated coverslips, responded to repeated addition of a suspension of unstimulated platelets by transient increases in cytosolic [Ca2+]i. These platelet-evoked Ca2+ responses were not caused by products secreted from the platelets and were insensitive to inhibitors of platelet activation and/or platelet aggregation. The platelet-evoked rises in [Ca2+]i in endothelial cells, similarly as the thrombin-evoked rises, were blocked by preincubation of HUVEC with the phospholipase C inhibitor U73122 or the Ca2+ influx blocker Ni2+. In contrast, treatment with the protein tyrosine kinase inhibitor genistein was without effect. Video imaging experiments, in which the fluorescence signal was analysed from the individual cells of an endothelial monolayer, showed that only 2-20% of the cells, scattered over the monolayer, responded to the addition of platelets by a transient increase in [Ca2+]i, whereas most of the cells responded to thrombin. This leads to the conclusion that unstimulated platelets can activate HUVEC putatively by mechanical interaction with individual endothelial cells in the monolayer.

Differential release of histamine and prostaglandin D2 in rat peritoneal mast cells: roles of cytosolic calcium and protein tyrosine kinases.

We studied how the release of histamine and prostaglandin D2 (PGD2) were connected in stimulated rat peritoneal mast cells, and to what extent these processes were controlled by the cytosolic Ca2+ concentration, [Ca2+]i, and protein tyrosine kinases. In the presence of 1 mM CaCl2, the G-protein activating compound 48/80 (10 micrograms/ml) evoked a transient rise in [Ca2+]i and a relatively high secretion of histamine, but only a low release of PGD2. In contrast, 5 microM thapsigargin (an inhibitor of endomembrane Ca(2+)-ATPases) and 5 microM ionomycin evoked high and prolonged rises in [Ca2+]i, and stimulated the cells to release relatively small amounts of histamine and high amounts of PGD2. Stimulation of the cells with CaCl2 and 10 microM ATP4- gave only minor quantities of histamine and PGD2, despite of the micromolar level of [Ca2+]i reached. When CaCl2 was replaced by EGTA, rises in [Ca2+]i as well as release of histamine and PGD2 were reduced with each agonist, but the preference of agonists to release more histamine or PGD2 remained unchanged. In mast cells with depleted Ca2+ stores, the addition of CaCl2 stimulated the store-regulated Ca2+ entry resulting in a prolonged rise in [Ca2+]i. However, simultaneous addition of compound 48/80 and CaCl2 was required for release of histamine and PGD2. In cells with full stores, PGD2 release evoked by compound 48/80 was greatly reduced by genistein and methyl-2,5-dihydroxycinnamate, two structurally unrelated inhibitors of protein tyrosine kinases, whereas histamine secretion was not influenced by these inhibitors. Similarly, with thapsigargin or ionomycin as agonist, PGD2 release was more sensitive to the tyrosine kinase inhibitors than histamine secretion. We conclude that in activated rat peritoneal mast cells: (i) the influx of extracellular Ca2+ potentiates agonist-evoked rises in [Ca2+]i as well as histamine secretion and PGD2 release; (ii) the amplitude of the [Ca2+]i rise does not determine the preferential effect of agonists to release more histamine or more PGD2; (iii) the relatively high PGD2 release evoked by thapsigargin and ionomycin is probably due to their potency to evoke a prolonged rise in [Ca2+]i and to activate protein tyrosine kinases.

Expression of transient receptor potential mRNA isoforms and Ca(2+) influx in differentiating human stem cells and platelets.

Store-regulated Ca(2+) entry (SOCE) is an important mechanism of elevating cytosolic [Ca(2+)]i in platelets, though the Ca(2+) influx channels involved are still unclear. We screened human platelets and their precursor cells (human stem cells and megakaryocytes) for the presence of candidate influx channels, i.e., isoforms of the Trp family of proteins. Primary stem cells were cultured with thrombopoietin to allow differentiation into megakaryocytes. The undifferentiated stem cells (CD34(+)) showed mRNA expression of only a spliced variant Trp1A. Immature (CD61(+)/CD42b(low)) and mature (CD61(+)/CD42b(high)) megakaryocytes as well as platelets expressed in addition unspliced Trp1 as well as Trp4 (less abundant) and Trp6 isoforms. This unspliced isoform appeared to be specific for cells of the megakaryocyte/platelet lineage, since immature (CD14(+)/CD61(-)/CD42b(-)) and mature monocytes expressed only the Trp1A isoform. This conclusion was confirmed by the presence of Trp1A, 3, 4 and 6 transcripts in the immature megakaryocytic Dami cell line, and of Trp1, 1A, 4 and 6 transcripts in the more mature CHRF-288 cell line. The up-regulation of Trp1, 4 and 6 in the lineage from primary stem cells to mature megakaryocytes and platelets was accompanied by increased influx of extracellular Ca(2+) after pretreatment of the cells with thapsigargin or thrombin. Expression of new Trp isoforms in the differentiated cells is thus accompanied by increased SOCE.

Nebivolol: a third-generation beta-blocker that augments vascular nitric oxide release: endothelial beta(2)-adrenergic receptor-mediated nitric oxide production.

BACKGROUND: Nebivolol is a beta(1)-selective adrenergic receptor antagonist with proposed nitric oxide (NO)-mediated vasodilating properties in humans. In this study, we explored whether nebivolol indeed induces NO production and, if so, by what mechanism. We hypothesized that not nebivolol itself but rather its metabolites augment NO production. METHODS AND RESULTS: Mouse thoracic aorta segments were bathed in an organ chamber. Administration of nebivolol did not affect NO production. When nebivolol was allowed to metabolize in vivo in mice, addition of plasma of these mice caused a sustained 2-fold increase in NO release. Interestingly, coadministration of a selective beta(2)-adrenergic receptor antagonist (butoxamine) prevented the response. Immunohistochemistry and Western blot analysis demonstrated the presence of beta(2)- but not beta(1)-adrenergic receptors on endothelial cells. In the absence of calcium, metabolized nebivolol failed to increase NO production, suggesting a role for calcium-dependent NO synthase. With digital fluorescence imaging, a rapid and sustained rise in endothelial cytosolic free Ca(2+) concentration was observed after administration of metabolized nebivolol, which also was abrogated by butoxamine pretreatment. CONCLUSIONS: In vivo metabolized nebivolol increases vascular NO production. This phenomenon involves endothelial beta(2)-adrenergic receptor ligation, with a subsequent rise in endothelial free [Ca(2+)](i) and endothelial NO synthase-dependent NO production. This may be an important mechanism underlying the nebivolol-induced, NO-mediated arterial dilation in humans.

Recombinant factor VIIa enhances platelet adhesion and activation under flow conditions at normal and reduced platelet count.

BACKGROUND: Recombinant factor VIIa (rFVIIa), which was developed for treatment of inhibitor-complicated hemophilia, appears suitable as prohemostatic agent in other clinical disorders including patients with thrombocytopenia. It is generally accepted that rFVIIa functions by enhancement of thrombin generation at the site of injury. It is, however, unknown if and how this affects platelet adhesion and aggregation. OBJECTIVES: To determine the effect of rFVIIa-mediated thrombin generation on platelet adhesion and aggregation under flow conditions at normal and reduced platelet counts. METHODS: Washed platelets and red cells were combined to obtain plasma-free blood with different platelet counts. The reconstituted blood was perfused over a collagen- or fibrinogen-coated surface in the absence or presence of a thrombin generating system consisting of purified coagulation factors rFVIIa, factor (F)X and prothrombin. RESULTS: Addition of coagulation factors rFVIIa, FX and prothrombin to washed platelets and red cells enhanced platelet adhesion and aggregation to collagen and adhesion and spreading to fibrinogen at normal platelet count and at platelet numbers as low as 10 000 microL(-1). rFVIIa-mediated thrombin generation enhanced the activation state of platelets as measured by intracellular calcium fluxes, and enhanced the exposure of procoagulant phospholipids as measured by annexin A5 binding. CONCLUSIONS: Taken together, increased platelet adhesion and aggregation by rFVIIa-mediated thrombin formation may explain the therapeutic effects of rFVIIa in thrombocytopenic conditions and in patients with a normal platelet count by (i) enhancement of primary hemostasis and (ii) enhancement of procoagulant surface leading to elevated fibrin formation.

Platelet adhesion enhances the glycoprotein VI-dependent procoagulant response: Involvement of p38 MAP kinase and calpain.

In the final stages of activation, platelets express coagulation-promoting activity by 2 simultaneous processes: exposure of aminophospholipids, eg, phosphatidylserine (PS), at the platelet surface, and formation of membrane blebs, which may be shed as microvesicles. Contact with collagen triggers both processes via platelet glycoprotein VI (GPVI). Here, we studied the capacity of 2 GPVI ligands, collagen-related peptide (CRP) and the snake venom protein convulxin (CVX), to elicit the procoagulant platelet response. In platelets in suspension, either ligand induced full aggregation and high Ca(2+) signals but little microvesiculation or PS exposure. However, most of the platelets adhering to immobilized CRP or CVX had exposed PS and formed membrane blebs after a prolonged increase in cytosolic [Ca(2+)](i). Platelets adhering to fibrinogen responded similarly but only when exposed to soluble CRP or CVX. By scanning electron microscopic analysis, the bleb-forming platelets were detected as either round, spongelike structures with associated microparticles or as arrays of vesicular cell fragments. The phosphorylation of p38 mitogen-activated protein kinase (MAPK) elicited by CRP and CVX was enhanced in fibrinogen-adherent platelets compared with that in platelets in suspension. The p38 inhibitor SB203580 and the calpain protease inhibitor calpeptin reduced only the procoagulant bleb formation, having no effect on PS exposure. Inhibition of p38 also downregulated calpain activity. We conclude that the procoagulant response evoked by GPVI stimulation is potentiated by platelet adhesion. The sequential activation of p38 MAPK and calpain appears to regulate procoagulant membrane blebbing but not PS exposure.

Acute and persistent platelet and coagulant activities in atherothrombosis.

The potential relevance of murine atherothrombosis models for understanding human disease has been debated in the past. Despite this, in the last decade, many thrombosis studies with atherogenic Apoe(-/-) mice have been performed, which provide novel insight into the molecular mechanisms by which platelet and coagulation processes accomplish acute thrombus formation after plaque disruption in vivo. Support for these mechanisms has come from whole blood flow perfusion studies over plaque material in vitro, which are also reviewed in this study. The main plaque-derived triggers for thrombus formation appear to be collagen and tissue factor, next to bioactive mediators such as prostaglandin E2. The atherothrombotic process relies on collagen- and ADP-receptor-induced platelet activation as well as on thrombin/fibrin generation via the extrinsic and intrinsic coagulation pathways. Less is known of the persistent effects of a thrombus on atherosclerosis progression, but evidence suggests roles herein of activated platelets and ongoing thrombin generation.

Desmopressin treatment improves platelet function under flow in patients with postoperative bleeding.

BACKGROUND: Patients undergoing major cardiothoracic surgery are subjected to dilution, owing to massive fluid infusion and blood component transfusion. These patients may experience bleeding perioperatively, and are frequently treated with the endothelium-activating agent desmopressin. OBJECTIVES: To investigate the effect of desmopressin administration on von Willebrand factor (VWF)-dependent coagulant and platelet functions under flow conditions. PATIENTS/METHODS: Blood from 16 patients with postoperative bleeding was obtained before and after desmopressin treatment (0.3 µg kg(-1) body weight), and assessed for coagulant properties and platelet function. Furthermore, VWF antigen levels and multimer composition were determined in both samples. RESULTS: Desmopressin treatment did not change thrombin generation in plasma or whole blood thromboelasticity. Also coagulation factor levels (other than factor VIII) and coagulation times were unchanged, suggesting that desmopressin treatment did not have a major effect on the coagulant activity. On the other hand, desmopressin treatment raised the already high plasma levels of VWF from a median of 116 IU mL(-1) (interquartile range [IQR] 102-154 IU mL(-1) ) to a median of 160 IU mL(-1) (IQR 126-187 IU mL(-1) ) (P = 0.007), owing to accumulation of the high molecular weight VWF multimers. Furthermore, desmopressin treatment caused an increase in collagen-dependent thrombus formation and platelet phosphatidylserine exposure. Markers of thrombus formation correlated with the plasma levels of VWF. In vitro control experiments confirmed a major contribution of VWF to thrombus formation and procoagulant activity under conditions of blood dilution. CONCLUSIONS: Desmopressin treatment of patients with bleeding complications after cardiothoracic surgery induces the release of high molecular weight VWF multimers, which enhance platelet activation and thrombus formation under flow conditions.

Calcium influx evoked by Ca2+ store depletion in human platelets is more susceptible to cytochrome P-450 inhibitors than receptor-mediated calcium entry.

We have previously reported that a component of ADP-evoked Ca2+ entry in human platelets appears to be promoted following the release of Ca2+ from intracellular stores. Other agonists may employ a similar mechanism. Here we have further investigated the relationship between the state of filling of the Ca2+ stores and plasma membrane Ca2+ permeability in Fura-2-loaded human platelets. Ca2+ influx was promoted following store depletion by inhibitors of the endoplasmic reticulum Ca(2+)-ATPase, thapsigargin (TG) and 2,5-di-(t-butyl)-1,4-benzohydroquinone (tBuBHQ). Divalent cation entry was confirmed by quenching of Fura-2 fluorescence with externally added Mn2+. It has been suggested that cytochrome P-450 may couple Ca2+ store depletion to an increased plasma membrane Ca2+ permeability. In apparent agreement with this, Mn2+ influx promoted by TG and tBuBHQ, or by preincubation of cells in Ca(2+)-free medium, was inhibited by the imidazole antimycotics, econazole and miconazole, which inhibit cytochrome P-450 activity. Agonist-evoked Mn2+ influx was only partially inhibited by these compounds at the same concentration (3 microM). Econazole (3 microM) reduced the Mn2+ quench evoked by ADP by 38% of the control value and that evoked by vasopressin, platelet activating factor (PAF) and thrombin no more than 15% of control, 20 s after agonist addition. Stopped-flow fluorimetry indicated that econazole had no detectable effect on the early time course of agonist-evoked Mn2+ entry or rises in [Ca2+]i. These data confirm the existence of a Ca2+ entry pathway in human platelets which is activated by depletion of the intracellular Ca2+ stores. Further, the results support the suggestion that cytochrome P-450 may participate in such a pathway. However, any physiological role for the cytochrome or its products in agonist-evoked events appears to be in the long-term maintenance or restoration of store Ca2+ content, rather than in promoting Ca2+ influx in the initial stages of platelet Ca2+ signal generation.

The roles of P(2X1)and P(2T AC)receptors in ADP-evoked calcium signalling in human platelets.

The roles of P(2X1)and P(2T AC)receptors in ADP-evoked Ca(2+)signalling were investigated in fura-2-loaded human platelets. Desensitization of the P(2X1)receptor with the selective agonist, alphabeta-methylene ATP, reduced the integral of the ADP-evoked rise in [Ca(2+)](i)to about 90% of control; a reduction equivalent to the integral of the P(2X1)-evoked response alone. After elevating cAMP or cGMP levels using prostaglandin E(1)or sodium nitroprusside, prior P(2X1)desensitization reduced the integral of the ADP-evoked response to about 70% of control. This reduction was greater than the integral of the P(2X1)-evoked response alone under the same conditions, suggesting rapidly activated Ca(2+)entry via the P(2X1)receptor potentiates Ca(2+)responses evoked via the phospholipase C-coupled P(2Y1)receptor. The P(2T AC)receptor antagonist, AR-C69931MX, at a concentration completely inhibiting aggregation, did not significantly affect the initial peaks but caused a significant reduction in the integrals of the ADP-evoked rises in [Ca(2+)](i)to about 71% or 77% of controls in the presence or absence of external Ca(2+)respectively. This suggests that the main effect of lowering cAMP levels after inhibition of adenylyl cyclase via P(2T AC)receptors may be reduced Ca(2+)removal from the cytosol. These results indicate that both the P(2X1)and P(2T AC)receptors play a significant role in ADP-evoked Ca(2+)signalling in human platelets.