Platelet Alpha-granule Release Research Articles

Investigation of the Relevance of Platelet Alpha Granules for Postoperative Liver Regeneration in a Mouse Model of Partial Hepatectomy

Introduction: Platelet alpha granule release was previously associated to development of liver dysfunction after liver resection. However, the relevance of platelet degranulation in liver regeneration has not been elucidated thus far. Here we aimed to investigate differences in liver regeneration between mice lacking alpha granules and wild type individuals after partial hepatectomy (pHx).

Bruton’s Tyrosine Kinase Inhibitors Impair Fcγriia-Mediated Responses of Healthy Donor Platelets and Chronic Lymphocytic Leukaemia Derived Platelets to Bacteria

Platelets play a key role in innate immunity and can interact with bacteria through multiple molecular mechanisms. One key receptor for platelet-bacteria interactions is FcγRIIa, a low affinity IgG immune receptor found on the surface of platelets, which is associated with platelet aggregation, phagocytosis and the release of bactericidal substances and cytokines from alpha and dense granules. The signalling pathways that regulate FcγRIIa mediated platelet responses to bacteria are not fully understood. Downstream of the FcγRIIa receptor is the protein Bruton's tyrosine kinase (Btk) and potentially other Tec family kinases, yet the role of these kinases in platelet-bacteria interactions is unknown. Btk is a therapeutic target in chronic lymphocytic leukaemia (CLL) with inhibitors of Btk (iBtks) including ibrutinib and acalabrutinib. However, iBtks have off-target effects on platelets, inhibiting aggregation with associated haemorrhagic side effects. iBtk treatment is also associated with a higher incidence of infection in CLL patients. The effect of iBtks on platelet immune function has not been evaluated. We hypothesise that Btk has a role in platelet FcγRIIa signalling in response to bacterial agonists, and that iBtks inhibit such responses, contributing towards the increased risk of infection seen with such therapies in CLL. We show that ibrutinib and acalabrutinib inhibit healthy donor FcγRIIa-mediated platelet aggregation, alpha and dense granule release in response to incubation with Staphylococcus aureus and Escherichia coli, and also in response to FcγRIIa crosslinking with the monoclonal antibody IV.3 (anti-FcγRIIa). The observed lack of granule secretion will reduce the bactericidal substances released by the platelet, as well as the amount of cytokine and chemokine secretion limiting cross-talk with other immune cells. Phosphorylation of Btk at tyrosine 223 (a marker of Btk activation) was detected in response to FcγRIIa agonists, and was inhibited by both ibrutinib and acalabrutinib. Little is known about the effect of iBtk treatment on CLL-platelet responses to bacteria. CLL patients tend to be on concurrent medications for comorbid conditions that could alter platelet responses. We show that treatment-naïve and ibrutinib-treated CLL platelets have aggregation and alpha granule release to thrombin receptor activator peptide 6 and ADP comparable to healthy controls, indicating that CLL platelet responses to these FcγRIIa-independent agonists are normal. Moreover, platelets derived from iBtk naïve CLL patients aggregate normally to bacteria in the presence of autologous plasma. However, platelets from ibrutinib-treated CLL patients have significantly inhibited aggregation and alpha granule release in response to S.aureus, E.coli, and IV.3 crosslinking. Moreover, Btk is phosphorylated at Y223 in response to bacterial agonists in treatment-naïve, but not in ibrutinib-treated CLL platelets, highlighting the lack of Btk activation in iBtk-treated patients. An X-linked agammaglobulinaemia (XLA) patient with a known Btk loss of function mutation was examined to investigate if Btk is vital for the FcγRIIa pathway. XLA-derived platelets aggregated normally in response to multiple bacterial species, suggesting Btk is redundant in mediating platelet aggregatory responses to bacteria. These results suggest that other Tec family kinases might have a role in platelet FcγRIIa activation to bacteria, which could be affected by iBtk treatment too. In conclusion, we propose that iBtks impair the FcγRIIa pathway in platelets, reducing platelet-bacteria responses, possibly contributing to the increased risk of infections in observed in CLL. Disclosures No relevant conflicts of interest to declare.

Analysis of Platelet Alpha and Dense Granule Release Using Small Volumes of Blood

A significant limitation of the gold-standard platelet function test, light transmission aggregometry (LTA), is the relatively large volume of blood required, as well as need for a platelet count in the resulting platelet-rich plasma of not much less than 100,000/μL, thus potentially precluding this test in pediatric and thrombocytopenic patients. Flow cytometric assessment of platelet activation in diluted whole blood avoids these limitations, typically employing anti-CD62P (P-Selectin) binding reflecting alpha granule release and PAC-1 binding reflecting conversion of GPIIb/IIIa to its active conformation. We have recently reported the use of tandem mass spectrometry to assess platelet dense granule release of deuterated serotonin in comparably diluted whole blood samples (https://doi.org/10.1093/ajcp/aqz094). In the present study we examine the possibility of adapting and optimizing the luciferin-luciferase (L-L) approach often used together with LTA for the assay of platelet dense granule ATP release from 10-fold diluted whole blood in parallel with flow cytometric analysis. We optimized the L-L reaction with commercially available reagents and used a conventional lumi-aggregometer to measure real-time secretion of platelet dense granules following agonist stimulation in 10-fold diluted whole blood obtained from healthy adult volunteers, with or without specimen stirring at 1000 RPM. Direct comparison of optimized reagents to a commercial L-L reagent kit showed an improved lower limit of detection for exogenously added ATP by at least one order of magnitude. Initial studies were performed under non-stirring conditions. Dose-dependency of agonist was observed, with representative results for released ATP of 243.1 ± 29.7 pmol/107 platelets in response to 40 μM TRAP (thrombin receptor-activating peptide) and 18.9 ± 2.4 pmol/107 platelets in response to 1 nM convulxin (GPVI receptor activator) (mean ± SEM, N = 15). In contrast, virtually no ATP secretion was observed in response to 10 μM ADP or to 10 μg/mL collagen. Despite the quite low platelet count (as low as 11,000/μL to date) following the 10-fold dilution of whole blood, introducing 1000 RPM stirring to these samples now permitted robust ATP release in response to collagen as low as 2 μg/mL. There continued to be little or no ATP release, however, in response to 10 μM ADP even with the addition of stirring. Flow cytometry was used to interrogate agonist-stimulated alpha granule release and GPIIb/IIIa conformation change. Following incubations with agonist and fluorescently-labeled antibodies, the 10-fold diluted whole blood was further diluted an additional 10-fold, and gating based on light scatter and binding of anti-CD61 antibody used to identify individual platelets. Under non-stirring conditions, TRAP, ADP, and convulxin all produced strong P-selectin exposure, as well as conversion of GPIIb/IIIa to its active conformation. Analysis of samples that had undergone 1000 RPM stirring during incubation with agonist showed quite similar results in response to TRAP. While still strongly positive, the responses to ADP in the stirred specimens showed decreases typically of at least 50%. Without stirring, platelet responses to 2-10 μg/mL collagen were virtually absent, whereas in the stirred specimens significantly positive anti-CD62P and PAC-1 binding were now observed. The combined flow cytometric and L-L studies required <1 mL blood. The results demonstrate the dramatic differences observed in the release of platelet alpha and dense granules, depending upon conditions and assay systems employed. We have now shown that with agonist addition to the same 10-fold diluted whole blood samples employed in flow cytometric platelet analysis, dense granule release may also be assessed by modifications of the L-L methodology already employed for lumi-aggregation studies in many laboratories. We have additionally shown that by employing stirring of the specimen during incubation with agonist, collagen itself may be employed as platelet stimulus, rather than having to rely upon convulxin. The difference in requirements for alpha versus dense granule release, however, remains exemplified by ADP: While a potent stimulus of alpha granule release, ADP employed in the 10-fold diluted whole blood setting appeared incapable of producing significant dense granule release either in the absence or presence of sample stirring. Disclosures Wool: Diagnostica Stago: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Abstract 52: IkB Kinase beta is a Key Modulator of Platelet Function, and the Remodeling of CARMA1-Bcl10-MALT1 Complex Formation

Secretion plays an important role in platelet function, and hence the secretory machinery offers a unique target to modulate thrombogenesis. We previously established that IκB kinase (IKK)-β is phosphorylated upon platelet activation, regulates their SNARE machinery, and involves CBM (CARMA1, Bcl10, and MALT1) complex formation. However, the detailed role of IKKβ in platelet function, the mechanism by which it regulates CBM complex formation and downstream effector activation remain elusive. Using a knockout mouse model system ( IKK β flox/flox -PF4Cre), we first showed that IKKβ plays a vital role in thrombus formation, and that it does so, in part, by regulating platelet functional responses, including dense and alpha granule release, αIIbβ3 activation, and PS exposure. Furthermore, we observed defects in compound fusion, platelet spreading, actin remodeling, and clot retraction. To this end, under clot retraction conditions in the knockout platelets, 7S complex formation was found to be inhibited. In terms of its signaling, using knockout platelets, we observed that IKKβ is required for the recruitment of Bcl10/MALT1 to CARMA1 upon platelet activation, i.e., CBM complex formation, and that this was due to the defective phosphorylation of Bcl10 and the IKKγ polyubiquitination. In conclusion, our data shows that IKKβ is a key regulator of platelet activation, in vitro and in vivo , and the remodeling of the CBM complex. Furthermore, our findings indicate that inducible clustering of signaling mediators and the formation of higher-order multi-protein complexes (i.e., the CBM complex) is a dynamic process, that supports nonlinear signaling networks and is important for platelet activation.

Abstract 56: Platelet Specific Knockout of Glucose Transporter 3 Leads to Altered Metabolism and Decreased Platelet Activation

Patients with diabetes display increased thrombosis and platelet activation. In these disorders, the systemic milieu is characterized by multiple metabolic changes including increased glucose concentrations. Preliminary metabolomics analysis of platelets from patients with type 2 diabetes revealed an accumulation of glycolytic and TCA intermediates relative to healthy controls. Therefore we hypothesized that decreasing platelet glucose uptake would limit glycolysis thereby decreasing energy production and platelet reactivity. Platelets import glucose via two glucose transporters GLUT1 and GLUT3. GLUT1 is expressed on the plasma membrane and GLUT3 is expressed predominantly on alpha granule membranes (85%) and to a lesser extent on the plasma membrane (15%). To better understand the consequences of glucose metabolism on platelet function we generated a platelet specific knockout (KO) of GLUT3 using a Pf4 Cre recombinase transgenic mouse crossed to mice that harbor floxed GLUT3 alleles. Platelet glycogen content and glycolytic intermediates were significantly reduced in GLUT3 KO platelets compared to controls, and following mitochondrial uncoupling exhibited reduced glycolysis rates. Interestingly, under these conditions, mitochondrial maximal respiration was increased two-fold, with no change in mitochondrial density, or citric acid cycle intermediates. In vitro , GLUT3 deficient platelets display a 90% reduction of spreading on fibrinogen and collagen matrixes and significant reductions in CD62p surface translocation and GPIIbIIIa activation following stimulation with multiple agonists. Additionally makers of alpha granule release were significantly reduced. In vivo analysis of GLUT3 KO mice using a 10% ferric chloride model of arterial thrombosis and a tail-bleed model indicated no alteration in thrombosis between littermate controls and knockouts. However in a KBx/N model of rheumatoid arthritis GLUT3 KO mice exhibited significantly reduced disease severity. Together, these data indicate that GLUT3-mediated glucose uptake is essential for platelet activation, spreading and alpha granule release. GLUT3 modulates mechanisms that promote rheumatoid arthritis but not those that regulate in vivo thrombus formation.

P1299 : Platelet alpha-granule release in liver regeneration after hepatectomy

P1299 : Platelet alpha-granule release in liver regeneration after hepatectomy

Abstract 522: Specific Oxidized Phospholipids Modulate Platelet Reactivity In Vivo During Hyperlipidemia Via Cd36: A Mechanism Linking Hyperlipidemia, Oxidant Stress And A Pro-thrombotic Phenotype

Enhanced platelet reactivity is critical to the pathophysiology of occlusive arterial thrombotic disease. Despite the strong clinical associations between hyperlipidemia, a major risk factor for atherosclerosis, and a pro-thrombotic phenotype, the mechanisms responsible for enhanced platelet reactivity during hyperlipidemia remain unknown. Pro-atherosclerotic lipid abnormalities such as hypercholesterolemia are associated with both enhanced oxidant stress and generation of biologically active oxidized lipids, including potential ligands for the scavenger receptor CD36, a major platelet surface glycoprotein. Using multiple murine in vivo thrombosis models we now demonstrate that hyperlipidemic atherosclerosis prone mice (apolipoprotein E-deficient or LDL receptor-deficient) form occlusive intravascular thrombi faster than wildtype mice, and that genetic deletion of CD36 protects mice from hyperlipidemia-associated enhanced platelet reactivity and accompanying pro-thrombotic phenotype. Structurally defined oxidized choline glycerophospholipid molecular species that serve as endogenous high affinity ligands for CD36 are shown to : be markedly increased in plasma of hyperlipidemic mice; be elevated in plasma of subjects with low HDL levels; and to promote platelet activation and alpha-granule release via CD36 at pathophysiological levels. These studies thus demonstrate that platelet CD36 interactions with specific endogenous oxidized lipids play a heretofore unrecognized role in the well-known clinical associations between hyperlipidemia, oxidant stress and a prothrombotic phenotype.

Activation of G12/13 Pathway Is Essential for the Dense Granule Release in Platelets.

Platelet secretion is an important physiological event in hemostasis. A number of agonists such as thrombin and thromboxane A2 induce platelet secretion. ADP does not cause dense granule release in aspirin-treated platelets, although ADP induce platelet shape change, aggregation and alpha granule release through Gq and Gi pathways. The protease activated receptors 1 and 4, and the thromboxane receptor activate the G12/13 pathways in addition to the Gq pathways. We postulated that the platelet dense granule release reaction depends on both Gq and G12/13, and in the absence of signaling through either G protein abolishes secretion. In other words, co-stimulation of Gq and G12/13 pathways is a major requirement for dense granule release in platelets. We rationalize that because U46619 and thrombin can activate both Gq and G12/13, they cause platelet dense granule release, whereas ADP fails to cause platelet secretion from dense granules because it does not activate G12/13. As a first step towards testing this hypothesis, we supplemented ADP signaling in aspirin-treated platelets with selective activation of G12/13 pathways using YFLLRNP, a partial agonist of PAR-1. YFLLRNP selectively active G12/13 signaling pathway without activating Gq or Gi pathways at low concentrations. YFLLRNP (60 μM) or 2MeSADP (100 nM) failed to cause dense granule release. However, addition of YFLLRNP (60 μM) and 2MeSADP (100 nM) together caused dense granule release. We proceeded to confirm these results in Gq null mice by selectively activating phospholipase C (PLC), a downstream signaling molecule from Gq. In aspirin-treated Gαq knockout mouse platelets 80 μM m-3M3FBS, a direct PLC activator, causes calcium mobilization from intracellular stores and platelet shape change, but does not cause dense granule secretion. We have previously shown that Gq/PLC pathways downstream of ADP are not sufficient for aggregation and require concomitant signaling from Gi for platelet aggregation. Thus, lack of aggregation by PLC activation alone in G?q knockout mouse platelets is consistent with our previous observations. In G?q null mouse platelets, m-3M3FBS (80 μM) or AYPGKF (500 μM) alone failed to cause dense granule release. However, addition of m-3M3FBS (80 μM) and AYPGKF (500 μM) together caused dense granule release. In addition, consistent with our previous findings, co-stimulation of G12/13 pathways and Gi (AYPGKF + 2MeSADP) in G?q knockout mouse platelets caused aggregation, but failed to cause dense granule release. We conclude that supplemental signaling from G12/13 is required for Gq-mediated dense granule release and that ADP fails to cause dense granule release because the platelet P2Y receptors, although activate PLC, do not activate G12/13 pathways.

Von Willebrand factor (VWF)-dependent human platelet activation: porcine VWF utilizes different transmembrane signaling pathways than does thrombin to activate platelets, but both require protein phosphatase function

The interaction between von Willebrand factor (VWF) and glycoprotein (GP) Ib results in platelet agglutination and activation of many signaling intermediates. To determine if VWF-dependent platelet activation requires the participation of pivotal transmembrane signaling pathways, we analyzed VWF-dependent platelet activation profiles following inhibition of several transmembrane signaling intermediates. This was accomplished using porcine VWF, which has been shown to interact with human GPIb independently of shear stress or ristocetin. Platelet alpha (CD62) and lysozomal granule release (CD63), microparticle formation, and platelet agglutination/aggregation were evaluated. The ability of signaling inhibitors to prevent VWF-dependent platelet activation was compared to their ability to inhibit thrombin-dependent activation. The results demonstrate that VWF-dependent platelet activation can occur independently of the activities of protein kinase C (PKC), wortmannin-sensitive phosphatidylinositide 3-kinase, and phospholipase C, as well as independently of elevations in the concentration of intracellular calcium. In sharp contrast, these transmembrane signaling intermediates are required for thrombin-dependent platelet activation. In addition, thrombin-dependent but not VWF-dependent platelet activation was associated with elevations in the concentration of intracellular calcium under the conditions used. The family of signaling intermediates which appeared to be pivotal for both thrombin- and VWF-dependent platelet activation were the protein tyrosine phosphatases and the serine/threonine phosphatases. It is concluded that thrombin-dependent platelet activation relies on the activation of several transmembrane signaling pathways, whereas VWF-dependent platelet activation is dependent upon the activity of protein phosphatases. Inhibition of these phosphatases in vivo may provide a novel therapeutic approach for treating VWF-dependent platelet disorders such as thrombotic thrombocytopenic purpura or arterial thrombosis.

Porcine von Willebrand factor and thrombin induce the activation of c-Jun amino-terminal kinase (JNK/SAPK) whereas only thrombin induces activation of extracellular signal-related kinase 2 (ERK2) in human platelets.

The interaction of platelets with subendothelial von Willebrand factor (VWF), especially under high shear stress, is considered to be the first activation step which primes platelets for subsequent haemostatic events. The signalling cascade which results from the interaction of VWF and its receptor GPIbIX has only been partially defined. Mitogen-activated protein kinases (MAPKs) are a family of downstream transmembrane signalling serine-threonine kinases and have been demonstrated to be present and functional in platelets; these include the extracellular signal-related kinases (ERKs), c-Jun amino-terminal kinases (JNKs) and p38 MAPK. Previously, we showed that p38 MAPK was not required in VWF-induced human platelet activation. It is not known whether VWF-dependent platelet activation involves the activation of the JNK and ERK family of signalling molecules. This report demonstrates that porcine von Willebrand factor (pVWF) induced a sustained and stable JNK activation measurable by 1 min after activation. Thrombin also induced JNK activation assessed at 1 min after activation. In contrast to thrombin, pVWF did not induce ERK2 activation at any time point tested. To ensure that ERK activation was unnecessary for pVWF-dependent platelet activation, we functionally inhibited ERK-dependent signalling with PD98059, a potent and selective inhibitor of the MAP kinase kinase (MEK-1), which is the upstream kinase of ERK1 and ERK2. Although PD98059 inhibited ERK2 activation in platelets, it had no effect on pVWF- or thrombin-induced platelet alpha or lysozomal granule release, modulation of membrane glycoprotein CD41, microparticle formation, platelet shape change or platelet agglutination. It is concluded that pVWF and thrombin induced JNK activation, but whereas thrombin induced ERK2 activation VWF did not; functional ERK2 activity was also not required for pVWF- or thrombin-dependent platelet activation.

P38 MAPK is activated but not necessary in porcine von Willebrand factor‐dependent platelet activation

We have investigated the role of p38 mitogen-activated protein kinase (MAPK) in von Willebrand factor (VWF)-dependent platelet activation. The interaction of platelets with subendothelial VWF, especially under high shear stress, is considered to be the first activation step which primes platelets for subsequent haemostatic events. As a model of VWF-dependent platelet activation, porcine VWF was employed. Porcine VWF induced p38 MAPK activation by 1 min post-addition; assessed by phosphorylation of a recombinant p38 MAPK fusion protein substrate termed glutathione S-transferase-MAPK activated protein kinase-2. To determine if p38 MAPK was necessary for porcine VWF-induced platelet activation, we functionally inhibited p38 MAPK activity with SB203580 before exposure of the platelets to porcine VWF. Inhibition of p38 MAPK had no effect on VWF-induced platelet alpha or lysozomal granule release, expression of activated GPIIb IIIa, modulation of membrane glycoprotein CD41, expression of phosphatidylserine as assessed by annexin V binding, microparticle formation, or platelet agglutination. It was concluded that SB203580-inhibitable p38 MAPK activity induced by porcine VWF is not necessary for platelet activation.

Inhalation of nitric oxide inhibits ADP-induced platelet aggregation and alpha-granule release

To gather further information about the effects on blood platelet activation of in vivo exposure to nitric oxide (NO), platelet reactivity was studied in blood from healthy, non-smoking male volunteers before and after 30 min inhalation of 40ppm NO. Whole blood was stimulated in vitro with adenosine diphosphate or thrombin receptor activation peptide (TRAP-6). In an ex vivo perfusion model, non-anticoagulated blood was exposed to immobilised collagen at arterial blood flow conditions (2600 s -1 ). Blood samples from both the in vitro and ex vivo experiments were stained with fluorochrome-labelled Annexin-V and antibodies against CD42a, CD45, CD49b, CD61, CD62P and fibrinogen, and analysed with a three-colour flow cytometry technique. NO inhalation reduced the platelet activation response to adenosine diphosphate (ADP) stimulation by decreasing platelet-platelet aggregation, alpha-granule release and platelet-leukocyte conjugate formation. TRAP-stimulated platelet activation, collagen-induced platelet activation and thrombus growth was unaffected by NO inhalation. We therefore suggest an ADP receptor inhibitor mode of action of inhaled NO, selective on the newly suggested G protein- and phospholipase C-coupled P2Y1 receptor. Our results demonstrate that blood platelet activation in healthy subjects is modulated by inhalation of NO in therapeutically relevant doses, although the clinical impact of our findings remains unclear.

Fibronectin, platelet factor 4 and beta-thromboglobulin in endemic hepatosplenic schistosomiasis: relation to acute hematemesis.

Some platelet alpha-granule contents were assessed in parallel with other markers of hemostatic imbalance in 50 patients with hepatosplenic schistosomiasis (15 patients with compensated hepatosplenomegaly, 15 patients with advanced hepatic fibrosis and ascites and 20 patients during an acute attack of hematemesis from ruptured esophageal varices). Platelet factor 4 (PF4), beta-thromboglobulin (beta-TG), fibronectin (FN), prothrombin fragment 1 + 2, thrombin-antithrombin (TAT) complexes, fibrin degradation products (FbDP) and D-dimer were assessed in schistosomal patients compared to controls (15 healthy subjects). A significant increase in both thrombin (high TAT and prothrombin fragment 1 + 2 levels) and plasmin (high FbDP and D-dimer levels) generation was detected in decompensated patients establishing the presence of a steady state of low-grade disseminated intravascular coagulation, with and without overt bleeding, in these patients. A decrease in plasma FN concentration was found in diseased groups compared to controls. The reduction in plasma levels of FN paralleled the defective liver function and matched the relative decrease in tissue FN in liver specimens of decompensated patients suggesting that FN levels can be used to evaluate the pathological staging of the disease. A significant increase in beta-TG and PF4 levels was noted in decompensated patients with ascites and/or acute hematemesis compared both to controls and compensated patients reflecting platelet alpha-granule release and consequently increased in vivo platelet activation which may initiate and/or perpetuate the pathophysiological mechanisms of the hemostatic imbalance underlying the hemorrhagic diathesis in hepatosplenic schistosomiasis.

Nitroprusside Inhibition of Platelet Function Is Transient and Reversible by Catecholamine Priming

BACKGROUND The time course and reversibility of sodium nitroprusside's in vivo inhibition of platelet function are unclear. METHODS Platelet aggregation and P-selectin expression as measures of platelet dense and alpha-granule release, respectively, were examined before and after administration of sodium nitroprusside (18 mg) to human volunteers and in in vitro studies. Hypotension occurring with sodium nitroprusside administration was treated with intravenous crystalloid and/or phenylephrine. RESULTS Compared with preinfusion studies, platelet aggregation to epinephrine was significantly inhibited immediately and 4 min after discontinuation of the sodium nitroprusside infusion but returned to baseline at 8 and 12 min after discontinuing sodium nitroprusside. However, both dense and alpha-granule release to adenosine diphosphate after in vivo sodium nitroprusside were never significantly inhibited even at the time when sodium nitroprusside infusion was maximal. In contrast to our in vivo findings, in vitro incubation of platelet-rich plasma with sodium nitroprusside resulted in significant inhibition of dense and alpha-granule release to adenosine diphosphate. These in vitro inhibitory effects of sodium nitroprusside were reversed by pretreatment with epinephrine but not phenylephrine. CONCLUSIONS In normal volunteers, sodium nitroprusside inhibits platelet aggregation to epinephrine but not adenosine diphosphate; inhibition was reversed within 8-12 min after discontinuing sodium nitroprusside. Sodium nitroprusside in vitro inhibition of platelet function to adenosine diphosphate was reversed by epinephrine pretreatment. Because of the rapid reversibility of its antiplatelet effect, sodium nitroprusside may be clinically useful even when there is the potential for impaired coagulation.

Time course of platelet alpha granule release in acute myocardial infarction treated with streptokinase.

To determine the time course of platelet alpha granule release in patients with acute myocardial infarction treated with streptokinase. A prospective study. Coronary care unit. Nine with myocardial infarction treated with both streptokinase and aspirin, and nine with acute chest pain but without myocardial infarction, who were treated with aspirin only. All patients received 250 mg aspirin on admission and 150 mg once daily thereafter. All patients who fulfilled the indications for streptokinase received 1.5 megaunits, in a single infusion. After the initial medication, serial measurements of plasma beta thromboglobulin and plasma platelet factor 4 were performed at fixed intervals after the onset of chest pain. The primary endpoint sought was the peak value of beta thromboglobulin and platelet factor 4 in each individual. The median peak plasma beta thromboglobulin in the infarction group was substantially higher than in those without infarction, at 37 (range 12 to 210) v 15 (9 to 36) mg/litre, P < 0.01. The corresponding values for plasma platelet factor 4 were 4.6 (2.4 to 60.0) v 2.2 (< 2 to 8.5) mg/litre, P < 0.01. Increased values were seen only within the first 12 h after onset of chest pain, and after 12 h there was no difference between the patients with myocardial infarction and those without. Aspirin treatment did not abolish alpha granule release. In patients with acute myocardial infarction treated with streptokinase the content of the alpha granules is released within the first 12 h after the onset of chest pain. Aspirin apparently does not abolish this release.

Nicotine effects on eicosanoid formation and hemostatic function: Comparison of transdermal nicotine and cigarette smoking

Objectives. The purpose of this study was to examine the possible role of nicotine in enhancing coagulation and to assess the potential cardiovascular toxicity of transdermal nicotine therapy for smoking cessation.Background. Cigarette smoking increases the risks of acute coronary events. A likely contributing mechanism is activation of coagulation. The role of nicotine in enhancing coagulability has not been resolved.Methods. We compared in a crossover study the effects of cigarette smoking, transdermal nicotine and placebo transdermal nicotine, each for 5 days, in 12 healthy smokers.Results. Cigarette smoking increased the urinary excretion of 11-dehydro-thromboxane B2(reflecting thromboxane A2generation) and increased plasma concentration of the platelet alphagranule constituents, platelet factor 4 and beta-thromboglobulin, compared with placebo treatment, indicating in vivo platelet activation. Cigarette smoking was also associated with higher levels of fibrinogen in plasma. Transdermal nicotine produced plasma levels of nicotine in the same range as those during smoking but had no effect on thromboxane A2metabolite excretion, platelet alpha-granule release or plasma fibrinogen, compared with placebo. Excretion of 2,3-dinor-6-keto-PGF1α(reflecting prostacyclin generation) was not significantly influenced by any treatment. These results suggest that nicotine as such is not responsible for the platelet activation or elevation of plasma fibrinogen seen in smokers. However, we cannot exclude the possibility that intermittent bolus-like dosing of nicotine from cigarettes could have different effects from those produced by continually released transdermal nicotine. Other findings were that cigarette smoking and transdermal nicotine treatment were both associated with a higher white blood cell count compared with the placebo patch condition, suggesting a direct effect of nicotine to increase circulating white cells. Factor VII coagulant activity (VIIc) was significantly lower during cigarette smoking, than during either nicotine or placebo patch conditions.Conclusions. Transdermal nicotine has less effect on platelet activation and catecholamine release than does cigarette smoking, and its use in smoking cessation treatment of patients with coronary heart disease is likely to be safer than cigarette smoking.

Activation in stored platelet concentrates: correlation between membrane expression of P-selectin, glycoprotein IIb/IIIa, and beta-thromboglobulin release.

By using two distinct measurements of alpha-degranulation (surface P-selectin [alpha-granule membrane protein-140] expression and beta-thromboglobulin [beta-TG] release) and quantitation of glycoprotein (GP) IIb/IIIa surface density, stored platelet concentrates were evaluated to determine a) which method of measuring platelet alpha-granule release was more sensitive in detecting early platelet activation; b) whether Day 1 levels of activation predicted the extent of activation or cell lysis on Day 5 of storage; and c) whether changes in surface GPIIb/IIIa density were primarily dependent on platelet activation. By using samples from paired and unpaired units stored for 1, 3, and 5 days, four observations could be made. 1) A flow cytometric assay for the percentage of P-selectin-positive platelets was more sensitive for early detection of platelet activation than was measurement of beta-TG release. This finding was most likely due to enhanced sensitivity in detecting platelets that had undergone partial alpha-granule release. 2) Total P-selectin expression correlated with beta-TG release, which indicated that the extent of alpha-granule membrane fusion with the external platelet membrane was proportional to the amount of alpha-granule contents released into the supernatant. 3) All of the activation measurements on Day 1 predicted the activation values, but did not predict the degree of cell lysis (measured by lactate dehydrogenase discharge), on Day 5 of storage. 4) Surface GPIIb/IIIa density was increased on the subset of P-selectin-positive platelets as compared with the P-selectin-negative subset at all times during storage, but, within each subset, GPIIb/IIIa surface density did not significantly increase over the time of storage.

Evaluation of thromboxane production and complement activation during myocardial ischemia in patients with angina pectoris.

The complement system and arachidonic acid metabolites are involved in severe myocardial ischemia such as myocardial infarction. Furthermore, there is experimental evidence for C5a participation in thromboxane production. We examined whether C5a and thromboxane are produced during brief and reversible episodes of myocardial ischemia induced in patients with stable angina. Twenty-five patients underwent either atrial pacing or percutaneous transluminal coronary angioplasty associated with arterial and coronary sinus blood sampling. Rapid atrial stimulation of patients with effort angina caused significant ST segment depression (delta ST = -1.7 +/- 0.2 mm), decreased fractional lactate extraction (from +12.8 +/- 2.5% baseline to -13.7 +/- 4.6% at peak ischemia, n = 13, p less than 0.001), and increased coronary sinus plasma thromboxane B2 levels (from 345 +/- 85 pg/ml baseline to 1,684 +/- 64 pg/ml at peak ischemia, p less than 0.01). Changes of fractional lactate extraction correlated significantly with changes of coronary sinus plasma levels of thromboxane B2. There was no change of coronary sinus 6-keto-PGF1 alpha levels. Similar pacing of control subjects (n = 6) did not cause release of lactate or thromboxane. Seventeen other patients underwent exercise testing with noninvasive measurements of thromboxane and prostacyclin metabolites in urinary samples collected before and after the test. No detectable increase of urinary 11-dehydrothromboxane B2 was measured in patients with stable angina after exercise-induced myocardial ischemia. However, basal 11-dehydrothromboxane B2 levels were significantly higher in patients with angina (105 +/- 25 pg/mmol creatinine, n = 9) than in control patients (45 +/- 8 pg/mmol creatinine, n = 8, p less than 0.05 between groups). Coronary sinus plasma levels of the anaphylatoxin C5a always remained below 4 ng/ml in patients undergoing pacing. More severe myocardial ischemia after coronary angioplasty (percent lactate extraction decreased from +24.8 +/- 2.7% baseline to -41.6 +/- 22.4% at peak ischemia, p less than 0.05) was not associated with C3a or C5b-9 generation. In all patients, there was neither platelet sequestration nor platelet alpha-granule release (no changes of beta-thromboglobulin/platelet factor 4 levels) into the coronary sinus plasma. Patients with stable angina have chronically increased thromboxane synthesis as assessed by excretion of urinary metabolites. Thromboxane is acutely released into the coronary sinus during pacing-induced ischemia without significant intracoronary platelet aggregation. Complement does not appear to be activated in stable angina during brief and reversible episodes of myocardial ischemia and does not contribute to thromboxane production.

In vivo effects of nonionic and ionic contrast media on beta-thromboglobulin and fibrinopeptide levels

Nonionic contrast media are suggested to cause increased thromboembolism (in vivo), platelet aggregation and procoagulant effect (in vitro) as compared with ionic contrast media. To study these effects in vivo, 30 consecutive patients undergoing routine angiography were prospectively randomized to three groups of 10 patients each. Group A received diatrizoate (ionic, high osmolality), Group B ioxaglate (ionic, low osmolality) and Group C iohexol (nonionic, low osmolality). In vivo platelet alpha-granule release and fibrin-1 formation were measured by radioimmunoassay of beta-thromboglobulin and fibrinopeptide A in peripheral venous samples.Levels were estimated at three stages daring the procedure: before and after left ventriculography and after coronary angiography. No differences were noted (p = NS) when the ratios of beta-thromboglobulin and fibrinopeptide A were compared among the three groups. These data suggest that the newer nonionic contrast media do not demonstrate enhanced systemic platelet activation or fibrin formation as compared with standard ionic contrast media. However, larger randomized clinical studies are necessary to conclusively establish the suggested thromboembolic potential of nonionic contrast media.

5-day storage of single-donor platelets obtained using a blood cell separator.

Platelets were collected from normal donors via a blood cell separator (Fenwal CS-3000). Platelets were stored initially in two separate 1000-ml bags (average count per bag, 2.3 +/- 0.5 x 10(11)) in 100 ml of autologous plasma for 5 days. Little change in platelet count was noted after 5 days of storage; however, the white cell count fell from 5.1 +/- 1.7 x 10(9) at Time 0 to 3.4 +/- 2.3 x 10(9) per l at Day five. The initial lactate values were 32 +/- 11 mg per dl and rose to 165 +/- 28 mg per/dl by 5 days. Platelet aggregation was impaired both by the collection procedure and during storage: whereas the response to ADP of the donors' platelets before the procedure was 100 percent, samples taken from the product immediately after collection had only a 45 percent response, which fell to 12 percent by Day 5. Aggregation using epinephrine was similarly affected, with a 75 percent response after collection and 0 percent response by Day 5. The plasma beta-thromboglobulin (beta-TG) level was high, both after collection (5.0 micrograms/ml at Time 0) and after storage (11.0 micrograms/ml), indicating a considerable effect of collection on platelet alpha granule release. In vivo recovery of these platelets was very good at 67 +/- 6 percent, with an average survival of 7.3 +/- 1.4 days (multiple hit; n = 4).(ABSTRACT TRUNCATED AT 250 WORDS)