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Background: Natural killer (NK) and NK T (NKT) cells are important in innate immune defense. Their unequivocal identification requires at least four antigens. Based on the expression of additional antigens, they can be further divided into functional subsets. For more accurate immunophenotyping and to describe multiple expression patterns of leukocyte subsets, an increased number of measurable colors is necessary. To take advantage of the technologic features offered by slide-based cytometry, repeated analysis was combined with sequential optical-filter changing., Methods: Human peripheral blood leukocytes from healthy adult volunteers were labeled with antibodies by direct or indirect staining. Tandem dyes of Cy7 (phycoerythrin [PE]-/allophycocyanin [APC]-Cy7), Cy5.5 (PE-/APC-Cy5.5), and PE-Cy5 and the fluorochromes fluorescein isothiocyanate (FITC), PE, and APC were tested alone and in combinations. Optical filters of the laser scanning cytometer were 555 DRLP/BP 530/30 nm for photomultiplier tube (PMT) 1/FITC, 605 DRLP/BP 580/30 nm for PMT 2/PE, 740 DCXR/BP 670/20 nm for PMT 3/Cy5/APC, and BP 810/90 nm for PMT 4/Cy7. Filter PMT 3 was replaced for detection of PE/Cy5.5 and APC/Cy5.5 by 740 LP/BP 710/20 nm and the sample was remeasured. Both data files were merged into one to combine the different information on a single-cell basis. The combination of eight antibodies against CD3, CD4, CD8, CD14, CD16, CD19, CD45, and CD56 was used to characterize NK and NKT cells and their subsets., Results: In this way Cy5.5 is measurable at 488-nm and 633-nm excitation. Further, with the two different filters it is possible to distinguish Cy5 from Cy5.5 in the same detection channel (PMT 3). With this method we identified NK and NKT cells, subsets of NK (CD3-16+56+, CD3-16+56-, CD3-16-56+) and NKT (CD3+16+56+, CD3+16-56+) and their CD4+8-, CD4-8+, CD4-8- and CD4+8+ subsets., Conclusion: With our adaptations it is possible to discriminate tandem conjugates of Cy5, Cy5.5, and Cy7 for eight-color immunophenotyping. Using this method, novel rare subsets of NK and NKT cells that are CD4/CD8 double positive are reported for the first time., (Copyright (c) 2005 Wiley-Liss, Inc.)

Background: Controversial results have been reported concerning the ability of fibrinogen receptor antagonists (fibans) to induce conformational changes in the fibrinogen receptor after binding to it as the initial step of fibrinogen binding and platelet activation., Methods: Platelets in citrated whole blood were stained with several pairs of anti-glycoprotein (anti-GP) IIb-directed monoclonal antibodies conjugated to phycoerythrin (PE) or indirectly labeled with Cy5. Pairs of monoclonal antibodies that induced a high-fluorescence resonance energy transfer (FRET) efficiency served as tools to detect activation-dependent changes of GP IIb after addition of adenosine diphosphate and several fibans., Results: Using the combination of the clones 5B12-PE and P2-biotin/SA-Cy5, a concentration-dependent alteration of the GP IIb conformation was observed after addition of tirofiban, eptifibatide, and lotrafiban. Magnitude and kinetics differed among the investigated substances., Conclusion: The newly developed FRET assay allows the direct investigation of conformational changes of GP IIb after addition of platelet agonists or receptor ligands, as shown for three fibans., ((c) 2004 Wiley-Liss, Inc.)

Paroxysmal nocturnal haemoglobinuria (PNH) is a rare, acquired stem cell disorder, characterised by an abnormal susceptibility of red blood cells to complement induced lysis, resulting in repeated episodes of intravascular haemolysis and haemoglobinuria, thromboembolic events at atypical locations and, to a much lesser extent, bleeding complications. Platelet function is assumed to be abnormal, however, a defect has not yet been characterised and underlying mechanisms remain elusive. To explore these issues, we investigated platelet function in PNH patients using assays for clot formation under low and high shear force (thrombelastography and PFA100 device), adhesion to glass beads in native whole blood (Hellem method), aggregometry using various agonists (Born method), and flow cytometric assays for baseline and agonist-induced surface expression density of alpha-granule (CD62P) and lysosomal granule proteins (CD63), ligand binding to surface receptors (thrombospondin), and expression density of activation-induced neoepitopes of the fibrinogen receptor complex (PAC-1). Platelet PNH clone size determined by CD55 and CD59 labelling was compared to the clone sizes of granulocytes, monocytes, erythrocytes, and reticulocytes. A profound reduction of platelet reactivity was observed in PNH patients for all "global function" assays (clot formation, adhesion, aggregation). Platelet hyporeactivity was confirmed using flow cytometric assays. Whereas baseline levels of flow cytometrically determined platelet activation markers did not differ significantly between controls and PNH patients, agonist-induced values of all markers were distinctly reduced in the PNH group. Moreover, significantly reduced white blood cell counts (3.1/nl vs. 5.9/nl), haemoglobin values (9.5 vs. 14.3/g per dl), and platelet counts (136 vs. 219/nl) delineate profound tricytopenia in PNH patients. The fraction of particular cell types lacking the surface expression of GPI-anchored glycoproteins is referred to as the respective PNH clone; median PNH clone sizes of cells with short life spans (reticulocytes, platelets, granulocytes) was 50-80% of total cell populations compared to 20% of red blood cells. The results of our laboratory investigations show, that in PNH, reduced platelet counts coincide with reduced platelet reactivity. The foremost clinical complication in PNH, however, is venous thromboembolism, very probably induced by an activated and dysregulated plasmatic coagulation system. From these seemingly contradictory findings we infer, that part of the platelet hyporeactivity is probably due to reactive downregulation of platelet function in response to chronic hyperstimulation. The overall result is thought to be an unsteady balance, associated with thromboembolism in a larger proportion of patients, and with bleeding in a smaller proportion.

Platelet-leukocyte conjugates are increased in cardiovascular disease, but exercise is also able to trigger platelet-leukocyte formation in healthy subjects. The aim was to investigate the heterogeneity of platelet-leukocyte conjugate formations triggered by short term exercise. 18 healthy non-smokers underwent a 90 second maximal test on a SRM cycle ergometry system and a control experiment. Blood samples were taken after 30 min rest, immediately before and after, 15 min and 1 h after exercise. The different platelet-leukocyte conjugates were detected by flow cytometry via CD45, CD14, CD16, CD41, together with CD62P antibodies for the investigation of platelet activation in the conjugates. In addition, a stimulation of conjugate formation in vitro with 8 microM TRAP-6 was initiated. Immediately after exercise platelet-granulocyte (+24%), and -lymphocyte (+17%) conjugates were increased (p<0.01), while the platelet-monocyte conjugates (+40%) were enhanced (p<0.05) 15 min after exercise. The differentiation after stimulation showed that the regular (CD14(+)16(-); +32%) and mature (CD14(+)16(+); +35%) monocytes were both increased after exercise (p<0.01) but the regular monocytes were preferred (p<0.001) in platelet-monocyte conjugate formation. In addition, these conjugates revealed the highest CD62P expression. Maximal short term exercise is useful for the investigation of platelet-leukocyte formation; e.g., it could be shown, that regular monocytes may be preferred in conjugate formation and that these conjugates revealed the highest CD62P expression.

Paroxysmal nocturnal haemoglobinuria (PNH) is characterized pathophysiologically by intravascular lysis of blood cells and clinically by thromboembolic events, often atypical in localization. In this study, we examined the plasmatic coagulation system of PNH patients to investigate a potential relation between coagulation alterations and disease intensity (PNH clone size). We found evidence for both an increase in procoagulant and in fibrinolytic activity, resulting in increased fibrin generation and turnover. Whereas a positive association of the procoagulant potential with PNH clone size was notable, fibrinolytic activity showed an inverse association with clone size. As a possible cause, a growing impairment of fibrinolytic activation and/or an increasing displacement of fibrinolytic activity is assumed. These mechanisms are most likely caused by the detachment of the glycosyl-phosphatidyl-inositol-anchored urokinase plasminogen activator receptor from cell surfaces, causing a progressive resistance to fibrinolytic stimuli, together with a probable shift of the fibrinolytic potential from cell surfaces to soluble, circulating complexes, resulting in a cellular fibrinolysis-steal phenomenon. Together, these processes are accused of mediating an increased thrombophilic risk in PNH. As hereditary prothrombogenic defects were found more frequently in patients suffering ischaemic complications, genetic thrombophilia seems to confer an additional thromboembolic risk in PNH, and should therefore be screened for.

Prolactin is a newly recognized platelet coactivator that functions through potentiation of ADP-induced platelet activation. However, the possible association between hyperprolactinemia and venous thromboembolism (VTE) has not been systematically investigated up to now; prolactin signaling mechanisms in platelets still need to be elucidated. In this study, plasma prolactin levels in healthy subjects and patients with VTE were determined, demonstrating that patients with VTE and no other congenital risk factors had significantly increased plasma prolactin levels. Moreover, prolactinoma patients demonstrated a higher incidence of VTE than the general population. To elucidate the molecular mechanisms for the development of venous thrombosis, prolactin receptor signaling during platelet activation was investigated with a focus on ADP-stimulated G-protein-regulated signaling pathways. The short isoform of prolactin receptors was detected on platelets. Signaling through this receptor, although not directly linked to Gq-proteins, substitutes for Gq-protein regulated signaling pathways involved in platelet activation. We identified protein kinase C, a well-established signaling molecule in platelet activation, as a target molecule for prolactin signaling pathways in human platelets. Our findings indicate that hyperprolactinemia may be an important novel risk factor for VTE, suggesting that its thrombogenic effect may be mediated through enhanced platelet reactivity. Revealing the molecular mechanisms of prolactin signaling will allow the design of new antithrombotic therapies.

Severe haemophilia is a serious, haemorrhagic disorder of the plasmatic coagulation system. In this study we investigated, whether 'compensatory' activation of the platelet coagulation system occurs in this situation. Platelet function was investigated with aggregation, adhesion and flow cytometric assays. In addition, we performed clot and platelet plug formation tests and determined endogenous thrombin potentials in patients with severe haemophilia A or B; results were compared to those of healthy controls. Platelet aggregation in response to stimulation with ADP, ristocetin and epinephrine was similar in patients and controls; aggregation in response to collagen was reduced significantly in haemophiliacs. Flow cytometric analysis of P-selectin (CD 62P) and CD 63, of the conformationally changed GP IIb/IIIa with PAC 1 and of thrombospondin bound to CD 36 (GP IV) was performed at baseline and post stimulation. Baseline expression of all markers was similar in haemophiliacs and controls. After stimulation of the platelet thrombin receptors with the thrombin receptor activating peptide (TRAP) 6, the surface expression of all markers increased significantly; again, the expression was similar in haemophiliacs and controls. With thrombelastography and PFA 100 analysis, clot formation under low shear and platelet plug formation under high shear is measured. Both test results revealed a significantly reduced clot and platelet plug formation capacity in severe haemophiliacs. Our results did not reveal signs of enhanced platelet preactivation in haemophiliacs, indicating that baseline platelet reactivity in severe haemophilia remains in a neutral state, despite the severely haemorrhagic condition. As expected, both thrombin and clot formation capacities were impaired significantly in severe haemophilia. The reduced response to collagen-based platelet stimulation tests is indicative of a concomitant platelet function defect. This defect probably contributes to the intensity of bleeding events in patients with severe haemophilia.

To elucidate potential causes for differing bleeding phenotypes of haemophilic patients of identical degree of coagulation factor deficiency, we investigated 21 male patients with severe haemophilia. Median annual coagulation factor demand and the extent of haemophilic arthropathy were used to discriminate between intensely and less intensely haemorrhagic phenotypes. Haemophiliacs with a median annual coagulation factor demand of 800 IU per kg bodyweight or more and with three or more joints affected by haemophilic arthropathy represented the intensely haemorrhagic phenotype group; all other patients comprised the less intense group. The discriminator values represent the respective medians of the overall group. The results of activated partial thromboplastin time, endogenous thrombin potential, pro- and anticoagulant factor analysis did not differ between the two groups. Median tissue-type plasminogen activator concentration (TPA) was elevated significantly in haemophiliacs with an intensely haemorrhagic phenotype, as was the activity of the thrombin-activatable fibrinolysis inhibitor. Median activity of the plasminogen activator inhibitor 1 (PAI 1) and the concentration of TPA-PAI 1 complexes were increased to approximately double those in nonsevere haemophiliacs. Coexistent congenital thrombophilia was found significantly more often in the less intensely haemorrhagic group. Thus, increased stimulation of the fibrinolytic system was associated with a more intensely haemorrhagic phenotype in our patients. We hypothesize that ineffective haemophilic haemostasis in response to trauma evokes a protracted stimulation of the entire haemostatic system, including costimulation of fibrinolysis. The absence of coexistent congenital thrombophilia predisposes to excess stimulation of fibrinolysis, which cannot be downregulated effectively due to the dysfunctional intrinsic pathway. The association of a more intensely haemorrhagic phenotype with a paradoxical hyperstimulation of the fibrinolytic system resembles a vicious circle, where bleeding seems to cause predisposition to more bleeding.

Objective: Exposure of platelets to anaesthetic agents may alter their functional characteristics and in patients undergoing surgery or long-term sedation this may be clinically relevant. We investigated the influence of thiopentone and propofol on the expression of platelet membrane P-selectin, fibrinogen receptor, and leukocyte-platelet aggregates ex vivo., Methods: In patients undergoing surgery blood samples were taken from a peripheral vein cannula before induction of anaesthesia with propofol (3.0 mg kg (-1) BW) or thiopentone (7.0 mg kg (-1) BW) and five minutes later (n = 11 in each group). Surface expression of the fibrinogen receptor complex (GP Ib/IIIa), P-selectin, and the percentage of leukocyte-platelet aggregates were measured by flow cytometry after stimulation via the ADP or thrombin receptor pathway., Results: There was no significant difference in the expression of gpIIb/IIIa, P-selectin, and percentage of leukocyte-platelet aggregates in the thiopentone and propofol group after induction of anaesthesia., Conclusions: In clinically relevant concentrations thiopentone and propofol did not influence the surface expression density of fibrinogen receptors, P-selectin molecules, and the percentage of leukocyte-platelet aggregates ex vivo.

Fourteen patients with different types of von Willebrand disease (vWD) having acute bleeds or elective surgery were treated with Immunate(sound recording copyright sign), a double-virus inactivated factor VIII/von Willebrand factor (FVIII/vWF) concentrate. The concentrate was applied as a bolus or via continuous infusion. FVIII activity (FVIIIc), vWF antigen (vWF:Ag), ristocetin cofactor activity (vWF:RCo), collagen binding activity (vWF:CB), activated partial thromboplastin time (aPTT), and von Willebrand multimers (vW-multimers) were monitored for 48 hours. Pharmacokinetic analyses were performed. The clinical efficacy was rated excellent or good. Bleeding complications occurred in 3 patients due to an additional FXIII deficiency in one patient, to a surgically induced bleed in another patient, and a rather short substitution period in the third patient. There were no serious adverse experiences. One patient showed a phlebitic reaction at the site of venous access after more than 100 hours of continuous infusion, requiring a change to application via bolus.

Background: After coronary artery stent implantation patients are treated with the adenosine diphosphatase (ADP) receptor antagonist clopidogrel to prevent subacute stent thromboses. Today these patients initially receive a loading dose of 300 mg of clopidogrel to accelerate the complete drug effect. In the current study we investigated whether a higher loading dose can shorten the time until the maximum platelet inhibitory effect of clopidogrel is achieved., Methods: P-selectin expression of nonstimulated and ADP-stimulated platelets was flow cytometrically measured before the clopidogrel loading dose and on 3 consecutive days in 52 patients with coronary artery disease: 21 patients in group 1 received 300 mg of clopidogrel after stent implantation and 11 patients in group 2 received the higher 450-mg clopidogrel loading dose followed by a daily dose of 75 mg of clopidogrel for both groups. The control group consisted of 20 patients who were monitored over 2 days before coronary intervention. Soluble P-selectin levels in plasma were determined by an enzyme-linked immunosorbent assay., Results: Inducible P-selectin expression on ADP-stimulated platelets was significantly reduced (P =.05) on days 1 and 2 in patient group 2 (450-mg loading dose) compared with group 1 (300-mg loading dose). No influence of clopidogrel on the P-selectin levels in plasma was observed., Conclusions: In our study the application of 450 mg of clopidogrel as the loading dose in patients undergoing coronary stenting shortens the period until the maximum effect of the ADP receptor antagonist is achieved and thus may lead to a more successful prevention of subacute coronary stent thromboses.

Pregnancy (including puerperium) is a period of hypercoagulability and seems to be an independent major risk factor for venous thromboembolism (VTE). However, the basis of the increased risk of VTE in pregnancy and around delivery is unknown. We hypothesized that changes in PRL, which is a prominently increased hormone during pregnancy and lactation, might be involved in the activation of platelets. To investigate platelet functional abnormalities in pregnancy, we assessed the ADP-stimulated and nonstimulated P-selectin expression of platelets in 42 consecutive pregnant women, 22 normo- and hyperprolactinemic patients with pituitary tumors, and controls. In addition, the aggregation of platelets by human PRL in vitro was studied. We found a significant correlation between PRL values and ADP stimulation of platelets in pregnant women (r = 0.56; P < 0.0001) and patients with pituitary tumors (r = 0.57; P = 0.006). Hyperprolactinemic pregnant women or hyperprolactinemic patients with pituitary tumors revealed significantly higher ADP stimulation of platelets (P < 0.0001) than healthy controls or normoprolactinemic patients with pituitary tumors. These results were reconciled by increased in vitro stimulation and aggregation of platelets using human PRL. Our novel findings demonstrate that hyperprolactinemia causes increased platelet aggregation via ADP stimulation both in vitro and in vivo. Moreover, our data indicate that PRL may be a physiological cofactor of the delicate coagulation balance during pregnancy and puerperium that might explain the increased risk of VTE in pregnant women around delivery. Further studies of the interaction between PRL and platelets will clarify the clinical relevance of hyperprolactinemia as a potential risk factor for VTE.

History and Admission Findings: A 27-year-old woman presented with increased bleeding tendency and known oculocutaneous albinism. In addition to frequent nasal and gingival bleedings she had since childhood spontaneous extensive cutaneous bleedings. In addition she was extreme sensitive to UV light with visual disturbances. Bone-marrow biopsy when aged 9 years had revealed abnormal pigment-storing cells. Physical examination showed a very fair skin and hair, spontaneous nystagmus and extensive rash in the face., Investigations: Prothrombin, partial thromboplastin and thrombin times as well as fibrinogen level were normal. On re- admission bleeding time varied greatly, while platelet count was normal. Mean platelet volume was slightly reduced. Von Willebrand antigen, factor VIII:C, factor IX:C, factor XIII and alpha (2)-antiplasmin were all normal. Aggregometry indicated abnormal platelet function; there was reduced platelet aggregation after stimulation with adenosine diphosphate. Flow cytometry of whole blood demonstrated abnormal storage function of thrombocytic delta-granules. The underlying cause of the described symptoms was shown to be a gene defect in typical location on the long arm of chromosome 10., Diagnosis and Treatment: The triad of tyrosinase-positive oculocutaneous albinism, abnormal thrombocytic delta-granules and ceroid deposition in the cells of the reticuloendothelial system or the bone-marrow is called Hermansky-Pudlak syndrome (after those who first reported it). There is as yet no causal treatment. But the patient was advised to use a nasal spray of desmopressin for spontaneous or difficult to stop bleedings after minor injuries. Desmopressin i. v. should be given prophylactically before any operation.

Objectives: to examine coagulation, fibrinolysis, and platelet activity in patients with peripheral vascular disease (PVD)., Design: fifty consecutive PVD patients and 50 healthy volunteers. (Prospective comparative study.), Materials and Methods: P-selectin expression in non-fixed, whole blood was measured flow cytometrically on non-stimulated and ADP- and TRAP-6-stimulated samples. Plasma fibrinogen, von Willebrand factor (vWF), tissue plasminogen activator (tPA), and plasminogen activator inhibitor-1 were determined using standard techniques. Disease severity was stratified on the basis of the ankle-brachial pressure index (ABPI) and the angiographic data were assessed using the Bollinger score., Results: coagulation and fibrinolysis parameters as well as the P-selectin expression on both stimulated and non-stimulated platelets were significantly increased in patients vs controls (all p<0.01). The respective sensitivity and specificity were as follows: P-selectin expression (81%, 94%), vWF (72%, 86%), fibrinogen (64%, 98%), PAI-1 (44%, 90%), tPA (15%, 100%). P-selectin expression on TRAP-6-stimulated MP correlated with disease severity (r=0.40, p<0.01)., Conclusions: these findings support the concept of ongoing thrombogenesis in the subclinical progression of PVD and demonstrate the high diagnostic sensitivity of flow cytometric analysis of platelet activation.

Background: The finding that platelets of patients with peripheral occlusive arterial disease (POAD) circulate in an activated state prompted us to study platelet signal transduction. We hypothesised that platelet hyperreactivity is caused by changes in intracellular signalling., Experimental Design: a single blood sample was taken from the antecubital vein of each participant prior to the start of intravenous treatment with prostaglandins., Setting: patients were recruited from our inpatient Department of Cardiology and Angiology at the University Hospital., Participants: 15 hospitalised patients with symptomatic POAD were randomly selected. Patients receiving antiplatelet drugs and those with diabetes were excluded. The control group consisted of 15 healthy volunteers from the medical staff., Interventions: blood tests were performed on the day of admission before any therapeutic intervention., Measures: the platelet activation marker P-selectin was quantified on peripheral blood platelets before and after in vitro stimulation with platelet agonists (adenosine diphosphate, thrombin receptor activator peptide-6). The signal transduction cascade was also selectively blocked by preincubation with either: 1) forskolin, 2) phospholipase C inhibitor U-73122, or 3) bisindolylmaleimide., Results: A stronger inhibitory effect on ADP-stimulated platelets was seen in patients with U-73122, as indicated by a decrease in mean fluorescence intensity of 51% versus 34% in controls (p<0.0005)., Conclusions: Our findings support the assumption that changes in platelet signal transduction in POAD lead to platelet hyper-reactivity.

Platelet volume has been reported to be increased in vascular disease. Therefore, we studied the relationship of mean platelet volume and platelet count as well as flow cytometrically measured platelet size and platelet function in 50 patients with peripheral arterial disease and 50 healthy volunteers. Platelet activation was measured by P-selectin expression analysis on resting and on stimulated platelets, and the determination of platelet aggregates and platelet-derived microparticles using flow cytometry. P-Selectin expression on platelets was significantly elevated in patients suffering from peripheral arterial disease (all P<0.0001). Platelet aggregates (P<0.0001) and platelet-derived microparticles (P<0.0001) were significantly higher in the patient group compared with controls, whereas mean platelet volume and platelet count showed no significant differences. Platelet count was inversely related to mean platelet volume in patients and controls (r = -0.43, P<0.001). The present study supports the hypothesis of platelet hyperreactivity and circulating activated platelets in peripheral arterial disease. Mean platelet volume, and platelet count cannot be used as predictive markers for platelet activation in peripheral arterial disease patients.

Platelet activation and adhesion to endothelial cells and extracellular matrix proteins are crucial events in the development of arterial cardiovascular diseases. Platelet activation is initiated by stimulation of intracellular signaling cascades, including the p42 mitogen-activated protein kinase (MAPK) and p38 MAPK pathways, followed by major changes in the platelet cytoskeleton and expression and activation of platelet surface receptors, such as P-selectin (CD62P) and CD40 ligand (CD40L). Activated platelets directly bind to vascular endothelial cells via CD40L/CD40 interactions and induce inflammatory reactions that initiate or aggravate atherosclerotic lesions. The aim of this study was to investigate effects of two known platelet inhibitors-the cAMP-elevating prostaglandin E(1) (PG-E(1)) and the cGMP-elevating sodium nitroprusside (SNP)-on platelet p42 MAPK and p38 MAPK activation as well as on surface expression of CD62P and CD40L. MAPK activation was analyzed by Western blot experiments using phosphorylation-specific antibodies, and surface CD40L and CD62P expression was determined by flow cytometry analysis. PG-E(1) and SNP strongly inhibited p42 and p38 MAPK phosphorylation as well as CD40L and CD62P expression in response to thrombin, a thromboxane A(2) analog, and ADP. These data indicate that adenosine and guanosine 3',5'-cyclic monophosphate-dependent protein kinases not only inhibit platelet pathways leading to activation and aggregation, but also those resulting in enhanced surface expression of protein ligands involved in inflammation. Expression of CD40L and CD62P was found to be independent of MAPK activation, since it was not inhibited by specific MAPK inhibitors. Inhibition of platelet-induced inflammatory responses including CD62P- and CD40L-mediated interaction of platelets with leukocytes and endothelial cells, respectively, is suggested to be an important component of the long-term vasoprotective effects of cyclic nucleotide-elevating prostaglandins and NO donors.

Background: Dysbalance of the coagulation and fibrinolysis system was suspected to be a further risk factor for the progression of peripheral occlusive arterial disease (POAD). Reports on disturbed platelet function in advanced disease, however, were contradictory. Therefore, we studied haemostasis parameters and platelet function in symptomatic patients with peripheral arterial disease., Methods: 60 peripheral arterial disease patients hospitalised for invasive diagnostic procedures were included into this comparative study. Patients were clinically stratified according to the criteria for chronic limb ischemia (grade I: n=36; grade II: n=11; grade III: n=13). Plasma fibrinogen, antithrombin III, von Willebrand factor, tissue plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1) prothrombin time, and activated partial thromboplastin time were determined using standard methods. We measured flow cytometrically, the platelet activation marker P-selectin on nonstimulated, ADP- and TRAP-6-stimulated platelets. Angiographic data were assessed using the Bollinger score., Results: Plasma levels of the procoagulant proteins fibrinogen (grade I: 3.7/grade II: 3.9/grade m: 4.0 g/l) and vWF (158/156/178%) increased and of antithrombin III (109/103/102%) and the PAI-1/tPA ratio (5.2/5.0/4.1) decreased with progressive disease. Highest platelet activation levels were observed in the CLI grade II subgroup. A significant correlation of disease severity was seen with the ankle-brachial pressure index (p=0.006; r=0.39) and with the Bollinger score (p=0.002; r=-0.41)., Conclusions: Progressive peripheral obstructive arterial disease was associated with platelet hyper-reactivity, haemostatic dysbalance of pro- and anticoagulant proteins, and a counterregulatory increase of fibrinolytic activity. Therapeutic concepts should include these pathogenetic mechanisms.

Aprotinin reduces blood loss following cardiopulmonary bypass operations (CPB) by the prevention of hyperfibrinolysis. Its influence on circulating platelets is uncertain. In this prospective trial we investigated activation, adhesion, and aggregation receptors on the platelet surface in 20 patients who underwent elective coronary artery bypass grafting. These patients were randomly assigned to receive either a high dose of aprotinin or placebo. Flow cytometry was performed to determine platelet activation [P-selectin, glycoprotein (GP) 53], adhesive (GP Ib), and aggregatory (GP IIb-IIIa) receptors on circulating platelets, before, during, and after CPB. Aprotinin had neither a significant effect on platelet activation nor on adhesive and aggregatory receptors. Plasma levels of D-dimers were measured before and after CPB to assess fibrinolytic activity. D-dimers following CPB and chest tube drainage were significantly less in the aprotinin group. We conclude that aprotinin reduces blood loss by its effect on fibrinolysis but has no direct influence on platelet function.

History and Clinical Findings: A 58-year-old man was admitted after he was found to have a huge aneurysm of the thoracic aorta. 38 months previously he had first experienced subacute pain in the right thorax. After this the aneurysm gradually increased in size. On admission a pulsating mass, 20 cm in diameter, was obvious, having broken through the right ventral thoracic wall. It was causing venous inflow congestion in the upper part of the body. The patient's general condition was poor: he had marked orthopnoea., Investigations: Serological tests for syphilis gave the following results: VDRL test, 1:32; FTA-ABS test reactive; TPHA test, 1:8000; treponema-specific IgM negative. Computed tomography with contrast-medium injection exactly defined site and extent of the aneurysm. It started at the aortic root and extended to the aortic arch. Third-degree aortic valvar regurgitation was demonstrated on echocardiography. Pulmonary function tests indicated severe restrictive ventilatory abnormality with clearly reduced respiratory reserve., Treatment and Course: The symptoms improved after 2 weeks on oral medication of captopril (25 mg/d), furosemide (80 mg/d), spironolactone (100 mg/d), codeine phosphate (90 mg/d) and thick paraffin (20 mg/d as needed). The patient declined further treatment. He died 8 weeks later at home.

Objective: In a prospective study surface antigens associated with platelet activation, aggregation, and adhesion and the platelet volume were measured to investigate the mechanism of the platelet function defect of cardiopulmonary bypass (CPB)., Methods: Blood samples were obtained during cardiac surgery before and after heparinization, as well as during and following extracorporeal circulation. The expression of the platelet surface glycoproteins (GP) IIb-IIIa, Ib, 53, and the granule membrane protein (GMP) 140 were measured using flow cytometry in platelet-rich plasma (PRP) before and after in vitro stimulation with adenosine diphosphate. A full blood count including mean platelet volume (MPV) was taken., Results: Heparinization resulted in a significant increase of GP 53 and GMP 140 and a significant decrease of GP Ib expression. During and following CPB, GP IIb-IIIa and Ib were significantly decreased. Similarly, the expression of the activation markers was reduced significantly. The mean platelet volume decreased significantly from 8.6 +/- 0.7 fl at baseline to 7.9 +/- 0.8 fl at the end of the study period., Conclusion: Our data suggest that heparinization induces platelet activation. We assume that a loss of larger and more activated platelets from the circulation contributes substantially to the platelet function defect of CPB.

The detection and characterization of anti-platelet antibodies which are directed to HLA class I molecules or platelet-specific glycoproteins is of great importance in the diagnosis and treatment of thrombocytopenia. In this paper a simple and rapid flow cytometric assay for the epitope-specific characterization of anti-platelet antibodies is described using fluorescence resonance energy transfer (FRET). Patient platelets or test platelets preincubated with patient serum were analyzed for surface-bound immunoglobulins using R-phycoerythrin-conjugated polyclonal anti-human IgG antibodies (excitation 488 nm, emission 585 nm). In a second step, HLA class I structures, platelet-specific glycoproteins (gpIIb/IIIa, gpIb), and the Fc gamma receptor II were stained with murine monoclonal antibodies and Cyan 5-labelled polyclonal anti-mouse IgG antibodies (excitation 585 nm, emission 670 nm). Upon monochromatic fluorescence excitation with a 488 nm argon laser the efficiency of light transfer from R-phycoerythrin to Cyan 5 is a direct measure of the distance between the human platelet-bound antibody and the epitope detected by the murine monoclonal antibody (mab). The assay permits discrimination between human antibodies directed to different platelet-specific glycoproteins or HLA class I structures without interference from non-specific Fc gamma receptor-bound immune complexes and also between antibodies directed to different epitopes on glycoprotein heterodimers (e.g., gpIIb/IIIa).

After a ten-day treatment of 21 patients with low molecular weight dextran (MW = 40,000), a decrease in the plasma concentrations of total cholesterol of 25%, LDL-cholesterol of 32% and apoprotein B of 22% was observed. The HDL fraction only showed a reduction of 10%. These dextran effects were dose-dependent. During the post-treatment period of 20 days a continuous increase in the lipid parameters to the preinfusion values was demonstrated. Dextran caused stronger lipid changes in HLP patients than in normolipaemics.

The relative number of free amino groups on the surface of low density lipoproteins (LDL) was determined after the incubation with low molecular weight dextran (MW 40,000) in several concentrations by two different assays (fluorescamine, TNBS). A decrease of the detectable free amino groups of 40% was shown by both assays. After the incubation of lysine with dextran these changes could not be observed. These results are explained with the aggregation of LDL particles influenced by dextran.

The effect of dextran infusions on plasma levels of total cholesterol, LDL and HDL cholesterol, apolipoprotein B and AI and triglycerides is investigated within a patient group. Some plasma lipids and lipoproteins still remain significantly lowered after the end of the 10 days treatment period. Comparing the normolipidaemic with the hyperlipidaemic patients of type IIa and IIb the degree of alterations is different between these 3 groups.