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PubMed1998

Differences in platelet alpha-granule release between normals and immune thrombocytopenic patients and between young and old platelets.

The risk of serious bleeding in patients with immune thrombocytopenic purpura (ITP) appears to be less than in comparably thrombocytopenic patients with megakaryocytic hypoplasia. It has been proposed that this difference is due to enhanced hemostatic activity of young platelets, which are increased in the circulation during ITP. We examined alpha-granule release in reticulated platelets (RP), which are thought to be the youngest circulating platelets, and in older non-reticulated platelets (non-RP) in normal human controls and ITP patients. Normal controls had a mean RP of 7%, compared with 42% in ITP patients. The mean concentration of thrombin receptor agonist peptide (TRAP) causing 50% of control RP to express CD62P (EC50) was 0.82+/-0.08 microM (SEM), significantly higher than the TRAP CD62P EC50 for RP in ITP, 0.57+/-0.06 microM (p = 0.04). Similarly, the TRAP EC50 for non-RP in controls, 0.84+/-0.09 microM, was significantly higher than in ITP, 0.56+/-0.07 microM (p = 0.03), suggesting that all platelets in ITP have an enhanced alpha-granule threshold response to TRAP compared with controls, while RP and older platelets within each patient group have similar threshold sensitivities to TRAP. By contrast, high-dose TRAP caused RP to express twice as much mean and total CD62P as non-RP in both ITP patients and controls (p <0.05 for both comparisons). We conclude that compared with controls, all platelets in ITP are primed to undergo alpha-granule release to TRAP, while in both ITP and controls, the newly circulating, reticulated platelets have the potential to contribute greater amounts of CD62P surface ligand compared with older platelets (non-RP) after stimulation. Both the increased RP% and enhanced platelet response to agonist in ITP may contribute to maintenance of hemostasis despite thrombocytopenia.

Adult↗
PubMed2000

Role of the recombinant non-integrin platelet collagen receptor P65 on platelet activation induced by convulxin.

Convulxin (Cvx) isolated from Crotalus durissus terrificus venom selectively binds with a high affinity to platelets and induces platelet aggregation by a mechanism that resembles that induced by collagen. Taking advantage that P65 has been recently cloned and expressed as a recombinant soluble protein (rec-P65), we examined the role of this non-integrin collagen receptor in platelet activation induced by Cvx. Rec-P65 blocked platelet adhesion to collagen-coated surfaces and inhibited platelet aggregation and ATP secretion induced by type I collagen. On the other hand, rec-P65 did not inhibit platelet aggregation and ATP secretion induced by Cvx, and it did not affect platelet adhesion to Cvx. In addition, ligand-blotting indicated that the Cvx binding to the collagen receptor GPVI was preserved in the presence of rec-P65. These observations indicate that P65 does not play a significant role in platelet activation by Cvx; in contrast, platelet response to collagen involves multiple receptors.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1991

Platelet-activating factor (PAF) receptor antagonists inhibit arachidonic acid induced platelet aggregation in rabbit whole blood.

The potency of several platelet-activating factor (PAF) receptor antagonists was measured by observing their inhibitory effects against PAF induced platelet aggregation. Their selectivity was assessed by monitoring their effect on platelet aggregation induced by arachidonic acid (AA) and adenosine diphosphate (ADP). The antagonists inhibited platelet aggregation induced at the PAF EC50 (0.023 microM) with the following rank order of potency: WEB 2086 greater than WEB 2170 greater than SRI 64-412 greater than SRI 63-675 greater than BN 52021 greater than kadsurenone greater than SRI 63-441 greater than alprazolam. While the antagonists had no inhibitory effect at the EC50 for ADP (10 microM), they did inhibit platelet aggregation induced at the EC50 for AA (55 microM). However, there was considerable variability in the slope of the inhibitory response and the relative potency of each antagonist against PAF induced platelet aggregation as compared to AA induced platelet aggregation. The antagonist IC50 (microM) against PAF and AA were as follows, with those that showed significantly different (p less than 0.01) slopes indicated by an asterisk: SRI 63-441* (3.8, 15.1); SRI 63-675 (1.4, 36.2); SRI 64-412 (0.5, 10.5); BN 52021* (2.4, 58.9); kadsurenone* (2.8, 28.3); alprazolam* (10, 25); WEB 2086 (0.055, 0.220), and WEB 2170 (0.107, 0.534). Therefore, in rabbit whole blood the antagonists were potent, although not completely selective, inhibitors of PAF induced platelet aggregation. These results suggest that the mode of action of PAF and AA induced platelet aggregation may share some common features. However, since the slope of the inhibitory response against PAF and AA for some antagonists differed, mechanistic differences in their action appear to exist.

Adenosine Diphosphate↗
PubMed1995

1-O-hexadecyl-2-metoxy-glycero-3-phosphatidylcholine--a methoxy ether lipid inhibiting platelet activating factor-induced platelet aggregation and neutrophil oxidative metabolism.

Whether or not two alkylglycerols could initiate a functional response in human platelets or modify responses induced by platelet activating factor (PAF) was evaluated. It was found that 1-100 microM 1-O-hexadecyl-2-metoxy-glycero-3-phosphatidylcholine (Et-16-OCH3) induced platelet aggregation but 1-O-hexadecyl-sn-glycerol (chimyl alcohol; CA) did not. Et-16-OCH3-induced platelet aggregation was abolished by pretreatment with the PAF receptor antagonist WEB 2086. While CA had no effect on platelet aggregation induced by PAF, pretreatment with Et-16-OCH3 (0.1 microM or higher) significantly inhibited platelet aggregation induced by PAF, but had no effect on aggregation caused by ADP, thrombin or phorbol myristate acetate (PMA). A receptor binding study using radiolabelled [3H]WEB 2086 showed that Et-16-OCH3 exerts its actions through interaction with the PAF receptor. Moreover, Et-16-OCH3 inhibited neutrophil chemiluminescence responses induced by PAF, but not reactions to PMA or a formyl peptide. Finally, 1 microM Et-16-OCH3 induced a rise in the intracellular calcium concentration in platelets equal to that induced by PAF and also had an calcium ionophore-like effect at 100 microM. Thus, this study shows that Et-16-OCH3 is both a potent inducer of platelet aggregation and an inhibitor of PAF-induced platelet aggregation and neutrophil chemiluminescence, through interaction with the PAF receptor.

Azepines↗
PubMed1993

Functional studies on platelets of a patient with an acquired disorder of platelet function associated with autoantibodies against membrane glycoprotein IIB/IIIA complex.

Platelet functions have been studied of a 63 year old woman with a severe acquired thrombopathy. The platelets did not adhere to siliconized glass. Aggregation could not be induced by either ADP (1 microM) nor collagen (2 micrograms/ml), no release of serotonin was found under these conditions. Thrombin caused only a weak aggregation response. Quantitative analysis of platelet actin revealed a very low total actin content (473 micrograms/10(9) platelets) and an extremely low F-actin value (3% of total actin). Stimulation of platelets with 0.1 U/ml thrombin for 3 min resulted in an increase of only 5% F-actin, whereas ADP and collagen did not induce any actin polymerization. Ca2+ movement in the patient's platelets is severely impaired after ADP and collagen stimulation, whereas a normal Ca2+ movement was obtained by 0.1 U/ml thrombin. The inhibition of the functions of normal platelets (aggregation and actin polymerization) by addition of patient's serum (5-10% final concentration) points to receptor blockade by platelet autoantibodies in the patient's serum. The antibody was purified by adsorption on Protein-A-Sepharose. Addition of IgG-suspension (5% final concentration) to washed control platelets resulted in similar effects on aggregation and actin polymerization compared to the effects of patient's serum.

Actins↗
PubMed1990

Filtration. A method to prepare white cell-poor platelet concentrates with optimal preservation of platelet viability.

HLA alloimmunization is a major problem for thrombocytopenic patients receiving long-term platelet support. It is caused by white cells (WBCs) that are present as contaminants in platelet concentrates (PCs). Recent data have shown that filtration is an effective means to reduce WBC contamination, but it has little effect on the recovery of platelets. The present report evaluates two filters, a cellulose acetate (CA) filter requiring the inactivation of platelets with prostacyclin and a cotton wool (CW) filter requiring no platelet inactivation. The results show that, using fresh pooled PCs from six random donors, both filters reduce WBC contamination below 10(7) per PC, the likely threshold below which alloimmunization does not develop. With platelets stored for 2 to 3 days the efficacy of the CW filter decreases. Neither filter inflicts important damage to the platelets, as there is no considerable platelet activation or cell disruption. Moreover, PCs prepared by both filters show normal survival and effectively reduce the bleeding times. Thus, filtration of PCs results in platelets with optimal responsiveness both in vitro and in vivo.

Animals↗
PubMed2005

Platelet adhesion assay--a new quantitative whole blood test to measure platelet function.

At this time no practical laboratory method for the measurement of the current functional state of blood platelets is available. A new innovative platelet adhesion assay (PADA) is described here. With only a short time requirement and minimal equipment, the PADA provides quantitative measurements of platelet adhesiveness. Only 0.5 mL of freshly drawn citrated whole blood is needed, to which special polymer particles are added. A defined shear grade is induced by a short period of shaking the sample. Proteins of the blood sample, especially fibrinogen, and thereafter also activated platelets, bind to the specific polymer surface. Following platelet counts both in the sample and in a control (blood without particles), the adhesion index (AI) is calculated as a quantitative measure of platelet adhesiveness. In healthy volunteers, a mean AI of 52+/-12 was measured. AI was shown to be nearly independent of the number of platelets in the sample (100 to 350 k/ microL), the hematocrit (44 to 25%), and the fibrinogen content (1.5 to 5 g/L). Age of the volunteers had only a minor influence on the AI. PADA was shown to be a simple reliable laboratory method for the detection of disturbed platelet function. The test requires low analytical efforts compared with other diagnostic platelet function tests.

Adult↗
PubMed2001

Monitoring the effects of platelet glycoprotein IIb/IIIa antagonists with a microtiter plate method for detection of platelet aggregation.

Measurement of platelet aggregation in platelet-rich plasma (PRP) is a fundamental tool in platelet studies, despite the fact that the technique required for this is time-consuming, may need large volumes of blood, and require particular skill and special equipment. The use of a microplate reader seems useful to perform platelet aggregation more rapidly and with less material. So, the aim of the present study was to validate a simple and rapid method which enables performance of kinetic measurements of platelet aggregation directly in a microtiter plate reader. Platelet aggregation was carried out in 96-well, flat-bottomed microtiter plates. Samples of PRP (140 microl/well) were placed in a microtiter plate. Agonists (10 microl/well) were added using an electronic multichannel dispenser directly before the reading was started. Measurements of the optical density were performed at 650 nm using a THERMOmax microplate reader (Molecular Devices, Sunnyvale, USA). During the run time the plate was incubated at 37 degrees C and was mixed with the automix function of the reader. The technique was verified by comparing dose-response curves of platelet agonists and glycoprotein IIb/IIIa antagonists, obtained with the standard aggregometer and with the microtiter plate reader. Platelet aggregation in microtiter plates is simple and rapid. It offers the advantages of lowering the test volumes and the possibility to perform about 90 tests simultaneously. The method was successfully applied to measure platelet inhibition by glycoprotein IIb/IIIa antagonists.

Adenosine Diphosphate↗
PubMed1990

Role of platelet membrane glycoprotein IIb-IIIa in agonist-induced tyrosine phosphorylation of platelet proteins.

Treatment of platelets with thrombin was shown previously to induce rapid changes in tyrosine phosphorylation of several platelet proteins. In this report, we demonstrate that a variety of agonists which induce platelet aggregation also stimulate tyrosine phosphorylation of three proteins with apparent molecular masses of 84, 95, and 97 kD. Since platelet aggregation requires the agonist-induced activation of an integrin receptor (GP IIb-IIIa) as well as the binding of fibrinogen to this receptor, we examined the relationship between tyrosine phosphorylation and the function of GP IIb-IIIa. When platelets were examined under conditions that either precluded the activation of GP IIb-IIIa (prior disruption of the complex by EGTA at 37 degrees C) or the binding of fibrinogen (addition of RGDS or an inhibitory mAb), tyrosine phosphorylation of the 84-, 95-, and 97-kD proteins was not observed. However, although both GP IIb-IIIa activation and fibrinogen binding were necessary for tyrosine phosphorylation, they were not sufficient since phosphorylation was observed only under conditions in which the activated platelets were stirred and allowed to aggregate. In contrast, tyrosine phosphorylation was not dependent on another major platelet response, dense granule secretion. Furthermore, granule secretion did not require tyrosine phosphorylation of this set of proteins. These experiments demonstrate that agonist-induced tyrosine phosphorylation is linked to the process of GP IIb-IIIa-mediated platelet aggregation. Thus, tyrosine phosphorylation may be required for events associated with platelet aggregation or for events that follow aggregation.

Antibodies, Monoclonal↗
PubMed1996

PTCA: periprocedural platelet activation. Part II of the Duesseldorf PTCA platelet study (DPPS)

BACKGROUND: Percutaneous transluminal coronary angioplasty (PTCA) with its various manipulations could create a dangerous, sudden haemostatic response. This study was performed to investigate PTCA-induced periprocedural changes in platelet activation and its consequences. METHODS: Twenty-five consecutive patients admitted for elective PTCA were preclassified as having or not having circulating activated platelets. Blood samples were taken for platelet activation marker analysis before, six times during and 2 h after PTCA. Intravascular platelet activation was analysed by flow cytometry to measure activation-dependent surface markers thrombospondin, P-selectin (CD62) and lysosomal GP53 (CD63). RESULTS: PTCA was associated with a significant reduction of peripheral platelet count. The initiation of the PTCA procedure led to a significant loss of more than 50% of the degranulated, activated platelets. After PTCA, the number of degranulated, activated platelets uniformly increased. CONCLUSIONS: We conclude that PTCA can induce consumption, particularly of preactivated platelets, and lead to sustained platelet activation after the procedure. This might explain why preactivated patients are at increased risk of suffering periprocedural ischaemic events and why increased thrombogenicity favours acute flow disruption and the progression of coronary stenosis at the lesion site.

Angioplasty, Balloon, Coronary↗
PubMed1993

Ultraviolet-B irradiation of platelets induces a dose-dependent increase in the expression of platelet activation markers with storage.

Ultraviolet B (UVB) irradiation of platelet concentrates (PCs) has been proposed as a novel technology to prevent HLA sensitization. We have recently reported that 2 J/cm2 of UVB radiation abolishes alloreactive lymphocyte responses in vitro. In order to increase the efficacy of UV irradiation for the prevention of HLA sensitization, we exposed PCs to 4 or 8 J/cm2 of UVB and evaluated the effect of UV radiation on platelet integrity during storage. We report here that UV exposed platelets show a progressive increase in the expression of activation markers P-selectin (GMP-140; CD62) and LIMP-CD63 (GP-53; CD63) on the platelet membrane over time in a dose-dependent manner compared to age-matched controls. Platelet metabolism was also enhanced as evidenced by significant changes in lactate and pH during post-irradiation storage. Based on these findings we transfused PCs within 4 h after UV irradiation. PCs exposed to 4 J/cm2 showed normal post-transfusion recoveries and haemostatic functions, while poor platelet recoveries were found after administration of PCs exposed to 8 J/cm2. We hypothesize that the rapid expression of P-selectin on platelets exposed to the higher dose of UVB leads to an increased binding of these platelets to leucocytes in the circulation resulting in poor platelet recoveries.

Antigens, CD↗
PubMed1998

Antecedent aspirin therapy inhibits baseline platelet activity in patients presenting with acute myocardial infarction. The GUSTO-III Platelet Substudy.

Aspirin therapy and platelet inhibition reduce the risk for the development of acute myocardial infarction (AMI). However, the effects of aspirin on baseline platelet activity in patients presenting with AMI are not known. We determined the effect of long-term aspirin use on baseline platelet activity in patients presenting with AMI, enrolled in the GUSTO-III Platelet Study. Platelet characteristics were investigated by aggregometry, flow cytometry and enzyme-linked immunosorbent assay in 23 patients before thrombolysis. Sixteen AMI patients were aspirin free, and 7 patients were using aspirin (81-500 mg/daily). The aspirin-treated patients exhibited a mild but consistent reduction of platelet activity which reached significance for 5 microM (p = 0.02), and 10 microM (p = 0.01) adenosine diphosphate induced aggregation. The surface expression of P-selectin (p = 0.02) and PECAM-1 (p = 0.03) and the plasma level of soluble P-selectin (p = 0.02) were also reduced. As previously observed in normal humans and patients with stable coronary artery disease, long-term aspirin therapy is also associated with diminished platelet activation in patients presenting with AMI. Long-term aspirin therapy mildly reduces baseline platelet activity; however, this degree of relative platelet inhibition does not appear to be cardioprotective.

Adenosine Diphosphate↗
PubMed1999

Significance of platelet-derived microparticles and activated platelets in diabetic nephropathy.

We measured levels of platelet-derived microparticles (PMP), which have coagulative activity and are produced by platelet activation or physical stimulation, and CD62P/CD63-positive platelets in patients with diabetes mellitus to determine their clinical significance and effects on complications of diabetes including diabetic nephropathy. We also compared these levels before and after administration of the antiplatelet drug cilostazol. Plasma PMP and CD62P/CD63-positive platelet levels were significantly higher in patients with diabetes mellitus than normal controls. CD62P-positive platelet levels were significantly higher in patients with nephropathy than in patients without complications. After administration of cilostazol, PMP and CD62P/CD63-positive platelet levels were significantly decreased. The increases in platelet activity and its related procoagulant activity appear to account in part for the hypercoagulability observed in diabetes mellitus. Our findings suggest that activated platelets might play a role in the development of diabetic nephropathy. Furthermore, antiplatelet therapy with cilostazol for diabetic patients may be useful as antithrombin therapy including antiplatelet therapy, since it suppresses the production of intrinsic coagulants produced by platelet activation.

Antigens, CD↗
PubMed2004

The effect of recombinant IgG antibodies against the leucine-33 form of the platelet beta3 integrin (HPA-1a) on platelet function.

Recombinant HPA-1a antibodies with Fc, mutated to remove destructive effector functions, have been developed as a potential therapy for fetomaternal alloimmune thrombocytopenia (FMAIT), via blockade of binding of human HPA-1a polyclonal antibodies to fetal HPA-1a1b platelets. We have assessed the effect of the IgG1 HPA-1a antibody B2G1 and two mutated derivatives in various functional assays in resting and agonist-stimulated platelets of the three HPA-1 genotypes. With HPA-1a1b platelets (fetal genotype), the antibodies did not activate signalling or CD62P expression in resting platelets, did not change in vitro bleeding time (IVBT), and did not inhibit platelet adhesion to collagen in flowing blood. Adhesion of HPA-1a1b platelets to fibrinogen was reduced by 20%, and aggregation induced by ADP by 50%, but collagen-related peptide (CRP-XL)-induced aggregation was normal. With HPA-1a1a platelets, aggregation to both ADP and CRP-XL was inhibited, with total blockade of adhesion to fibrinogen and of IVBT responses. Interestingly, a monovalent antibody fragment with identical specificity had no inhibitory effect on aggregation. In static adhesion assays using human alphaIIbbeta3 or alphaVbeta3 transfectants of HPA-1a (Leu(33)) phenotype, attachment to fibrinogen of the latter but not of the former was completely blocked by the HPA-1a antibodies. These observations are best explained by antibody-mediated blockade of the RGD binding site on beta3 by a mechanism of steric hindrance. As the effect on platelet function is modest with HPA-1a1b (fetal type) platelets, the mutated HPA-1a antibodies described here could be developed further for FMAIT therapy.

Antibodies↗
PubMed1980

Biochemical studies of two patients with the gray platelet syndrome. Selective deficiency of platelet alpha granules.

The biochemistry of platelets from two unrelated patients with the gray platelet syndrome, a deficiency of platelet alpha-granules, has been evaluated. Ultrastructural studies of their platelets revealed the number of alpha-granules to be less than 15% of normal, whereas the number of dense bodies was within normal limits. Platelets from both patients had severe deficiencies of platelet factor 4 and beta-thromboglobulin (less than 10% of normal). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a marked deficiency of thrombin-sensitive protein in both patients. Analysis of the platelet-derived growth factor in one patient showed it was also markedly reduced. Levels of lysosomal enzymes, adenine nucleotides, serotonin, and catalase, and conversion of arachidonic acid by the lipoxygenase and cyclo-oxygenase enzymes, were within normal limits. The results provide important evidence to define the contents of alpha-granules and to differentiate these contents from the contents of lysosomal granules, dense bodies, and peroxisomes. Functional studies of these platelets showed deficiencies in ADP, thrombin, and collagen aggregation. The results suggest that alpha-granules or their contents make a contribution to normal platelet aggregation.

Acid Phosphatase↗
PubMed1990

Vitronectin binds to activated human platelets and plays a role in platelet aggregation.

Vitronectin (Vn) is a multifunctional 75-kD glycoprotein that is present in plasma and the extracellular matrix. Vn functions as a complement regulatory protein in plasma, and promotes the growth and attachment of cells in tissue culture. Recent cDNA cloning reveals that like other adhesive proteins, Vn contains the sequence Arg-Gly-Asp and binds to some members of the integrin class of adhesive membrane receptors. In liposomes, the platelet membrane glycoprotein complex IIb/IIIa binds Vn, as well as fibrinogen, von Willebrand factor, and fibronectin. We examined the binding of purified Vn to resting and stimulated human platelets. Vn bound to thrombin-stimulated platelets in a calcium-dependent, specific, and saturable manner with a Kd of 320 nM and 8,000 sites per platelet. Epinephrine or ADP stimulation led to specific binding with KdS of 93 and 116 nM, respectively. Binding was inhibited by the tetrapeptide Arg-Gly-Asp-Ser and by monoclonal and polyclonal antibodies to GPIIb/IIIa. Endogenous platelet Vn stores were identified in immunoblots of gel-filtered platelets and the surface expression of endogenous platelet Vn was thrombin inducible. Monoclonal as well as polyclonal antibodies to Vn inhibited platelet aggregation, suggesting that Vn plays a role in the formation of stable platelet aggregates.

Antibodies↗
PubMed1976

Bernard-Soulier syndrome: a new platelet glycoprotein abnormality. Its relationship with platelet adhesion to subendothelium and with the factor VIII von Willebrand protein.

A decreased platelet adhesion to rabbit aorta subendothelium (Baumgartner technique) is confirmed in the Bernard Soulier (giant platelet) syndrome. Electron microscope techniques using a purified antibody against Factor VIII/von Willebrand protein, revealed an apparently normal presence of the Factor VIII/von Willebrand protein on the Bernard Soulier platelets. Electrophoretic characterization of the major protein and glycoprotein components of the Bernard Soulier platelets following sodium dodecyl sulfate solubilization indicated a relatively normal protein content but suggested a reduced content of the 155,000 molecular weight major platelet glycoprotein. This was confirmed by a reduced release of high molecular weight acidic glycopeptides following incubation of washed Bernard Soulier platelets with trypsin. It is proposed that this abnormality may be related to the previously reported reduced sialic acid content and the reduced electrophoretic mobility of the Bernard Soulier platelets and that a glycoprotein reduced or abnormal in the Bernard Soulier platelets is necessary for the normal adhesion of platelets to subendothelium.

Adult↗
PubMed1982

Determination of the density distribution of human platelets--methodological aspects and comparison with other tests for platelet activation.

Stimulation of human platelets to release results in decreased buoyant density. This decreased density provides a tool to detect circulating platelets which have participated in a thrombotic process. Platelet density gradient centrifugation using Stractan was standardized and the effects of anticoagulation, temperature, and osmolarity were investigated. In 7 out of 32 patients with thrombotic disease less dense platelets were found. Platelet activation in the patient group was also indicated by spontaneous aggregation (10/32), decreased circulating platelet aggregate ratios (5/24) and elevated plasma beta-thromboglobulin levels (2/ 11). Several of these tests were also abnormal in diabetes mellitus thrombocytosis, leukaemia and several systemic diseases with thrombotic complications. The platelet density test using Stractan is reproducible and independent of other tests for platelet activation and is therefore potentially a useful extension of platelet function testing in patients with thrombotic disease.

Adult↗