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PubMed1975

An evaluation of the Coulter thrombocounter in counting platelets before and after contact with foreign surfaces and its use in tests of platelet retention.

The counting of platelets by electronic methods has become widely accepted by laboratories and the methods is considered to give better reproducibility than the conventional microscope count. Prompted by an impression that prior contact of whole blood with glass beads or cellulose-based dialysis membranes resulted in platelet counts showing a poor correlation between the electronic and microscopic methods of counting, an evaluation of the Coulter Thrombocounter was carried out with regard to its ability in counting platelets before and after contact with such foreign surfaces in two tests for the measurement of platelet retention. The effect of citrate and heparin anticoagulation with and without the addition of EDTA on the efficiency of the Thrombocounter was also studied. The results indicate that an excellent correlation is obtained between microscopic and electronically estimated platelet counts before foreign surface contact when EDTA is included in the anticoagulation, and that the Thrombocounter gives good reproducibility over a wide range of platelet counts. After contact with the foreign surfaces, the Thrombocounter significantly underestimated the platelet counts and thus overestimated the platelet retention values. Using the membrane test cell system with citrate plus EDTA anticoagulation, platelet retention results estimated by Thrombocounter platelet counts were significantly higher than those estimated by microscopic platelet counts but there was a good linear correlation between the two sets of results. Thus, the Thrombocounter may be used with this method. However, using the glass-bead column technique and the same anticoagulation, although the platelet retention results gave the same mean value with both methods of platelet counting, there was no correlation between the two sets of results indicating that microscopy alone should be used with this method. The other anticoagulant regimens rendered the Thrombocount inefficient for use in these tests of platelet retention.

Blood Cell Count↗

PubMed1994

Complement activation in platelet concentrates is surface-dependent and modulated by the platelets.

Activation of platelets during storage may contribute to the loss of function and viability known as the platelet storage lesion. We investigated the role of complement activation as a possible mediator of the platelet storage lesion. We studied platelet bags stored up to 5 days under standard blood bank conditions and monitored the generation of several complement activation fragments from both the classical and alternative pathways. To assess the role of noncellular artificial surfaces in the activation of complement, parallel bags were prepared containing platelet-poor plasma. The levels of C4d and C3a increased steadily over time in storage, as did the level of the inactivated membrane attack complex SC5b-9. Generation of C4d, C5a, and SC5b-9 was greater in the absence of platelets than when platelets were present in the container. C5a levels in both groups were low and remained so during storage, suggesting that the C5a generated became surface associated. Using flow cytometry we detected C3 and C3a, but not C9, on the platelet surface. The percentage of C3-positive platelets peaked at the third day of storage; by day five platelet-associated C3 had declined. Decay accelerating factor expression on the platelet surface increased with time in storage and in parallel with CD63 expression. Based on the C4d levels, complement activation proceeded via the classical pathway; minimal generation of the alternative pathway activation fragment. Bb was seen in either the presence or absence of platelets. As an indirect measure of the activation of C1, the functional level of C1 esterase inhibitor (C1INH) was determined. C1INH levels declined over time in storage in bags containing only plasma; however, in the presence of platelets, the levels remained constant presumably because of release of C1INH from the alpha-granules of activated platelets.

Blood Platelets↗

PubMed1984

Effect of epinephrine on fibrinogen receptor exposure by aspirin-treated platelets and platelets from concentrates in response to ADP and thrombin.

Synergistic effects between agonists on platelet aggregation have long been appreciated. Recently epinephrine was reported to induce maximal aggregation of aspirin-treated platelets when combined with ADP or thrombin, and to increase fibrinogen binding of non-aspirin treated platelets stimulated with low doses of ADP. The present study extends these observations to correlate fibrinogen binding in response to various combinations of ADP, epinephrine, and thrombin with platelet aggregation and 14C-serotonin release using aspirin-treated platelets as well as platelets from stored concentrates. When fresh platelets were stimulated with epinephrine (5 microM) together with either ADP (10 microM) or thrombin (150 mU/ml), fibrinogen binding increased by 180% compared to binding observed in response to ADP or thrombin alone. This was accompanied by enhanced platelet aggregation, but no increase in 14C-serotonin release. While both ADP and epinephrine potentiated the aggregation and fibrinogen binding of stored platelets in response to high doses of thrombin (150 mU/ml), maximal aggregation was achieved only with thrombin (150 mU/ml) and epinephrine (5 microM) in combination. The data thus suggest that 1) epinephrine induces maximal aggregation of aspirin-treated platelets stimulated with thrombin or ADP by significantly enhancing fibrinogen receptor exposure independently of the cyclooxygenase-mediated release reaction; 2) epinephrine stimulates platelets by a mechanism different from that of thrombin or ADP; and 3) as demonstrated by others, the ability of platelets from stored concentrates to aggregate and to bind fibrinogen in response to ADP can be enhanced by epinephrine, and, in addition, these platelets can aggregate and bind fibrinogen maximally when stimulated with combinations of epinephrine and thrombin.

Adenosine Diphosphate↗

PubMed1986

Potentiation by adrenaline of human platelet activation and the inhibition by the alpha-adrenergic antagonist nicergoline of platelet adhesion, secretion and aggregation.

Adrenaline (1 to 10 microM) can induce the aggregation of human platelets suspended in citrated plasma but does not induce the aggregation of washed human platelets at doses as high as 1 mM, although these platelets respond normally to ADP, PAF-acether, collagen, arachidonic acid, thrombin, the endoperoxide analog U-46619 and the Ca2+ ionophore A23187. Adrenaline (0.5 microM) potentiates the aggregation and secretion induced by all the previous agonists in citrated platelet-rich plasma (cPRP) or in washed platelets. The activation by adrenaline of human platelets is mediated by alpha 2-adrenergic receptors, as demonstrated by inhibition with a series of adrenergic antagonists. The alpha-adrenergic antagonist nicergoline inhibits the activation of human platelets by adrenaline in the following situations: nicergoline inhibits the aggregation and secretion caused by adrenaline in cPRP (IC50 0.22 microM and 0.28 microM respectively); nicergoline inhibits the aggregation and secretion induced by the combination of adrenaline and each aggregating agent listed above in cPRP (IC50 ranging from 0.1 to 2.5 microM) or in washed platelets (IC50 ranging from 0.1 to 0.8 microM); nicergoline inhibits the binding of 3H-yohimbine to washed human platelets (IC50 0.26 microM); the intravenous administration of nicergoline (0.5 mg/kg per day) to patients inhibits significantly the ex vivo response of their platelets to adrenaline in cPRP. High concentrations of nicergoline also inhibit the aggregation and secretion induced by the aggregating agents listed above in cPRP (IC50 range 108 to 670 microM) and in washed platelets (IC50 range 27 to 140 microM) and the adhesion of platelets to collagen-coated surfaces. This latter effect is not mediated through blockade of alpha-adrenoceptors. A possible role of adrenaline in platelet activation in vivo could justify the use of nicergoline (Sermion), an alpha-adrenergic antagonist in combination therapy to prevent arterial thrombosis.

Blood Platelets↗

PubMed1987

Effect of the amount and type of dietary fat on platelet function, platelet survival and response to continuous aortic injury in rats.

The effect of giving diets containing 1.5 or 16% safflower or corn oil or 16% milk fat for 15 weeks on changes in the fatty acid composition of platelet phospholipids, in vitro platelet function, platelet survival and thrombosis was examined in rats. The mean plasma cholesterol concentration was not different among the groups. Diets containing 1.5% safflower or corn oil or 16% milk fat were associated with a decrease in 18:2n - 6 and an increase in 18:1n - 9 and the 20:4n - 6/18:2n - 6 ratio in the platelet phospholipids compared with the 16% safflower or corn oil diets. The 16% milk fat diet was associated with an increase in 14:0, 20:3n - 9, 22:3n - 9 and a decrease in 22:4n - 6 in platelet phospholipids compared with the other groups. There were no differences among the groups in the sensitivity of washed platelets to ADP-, thrombin- or collagen-induced aggregation, or thrombin- or collagen-induced release of granule contents or loss of arachidonate from platelet phospholipids. Platelet survival and turnover in rats given the diets were not different among the groups. In response to indwelling aortic catheters neither the percentage reduction in platelet survival nor the platelet accumulation on injured aortae and catheters were different among the groups. No macroscopic thrombi were seen in rats given any of the diets. The results of these studies provide no evidence that diet-induced alterations in fatty acid content (increases in 18:1n - 9, 20:3n - 9, 22:3n - 9, 20:3n - 6, and 20:4n - 6/18:2n - 6 ratio and a decrease in 22:4n - 6) of platelet phospholipids modify in vitro platelet function, platelet survival or turnover or influence thrombosis in rats.

Animals↗

PubMed1991

Platelet hypersensitivity in cholesterol-fed rabbits: enhancement of thromboxane A2-dependent and thrombin-induced, thromboxane A2-independent platelet responses.

Hypercholesterolemia (mean plasma cholesterol: 15 mM) was induced in rabbits by the feeding of a chow diet enriched with a low amount (0.25%, w/w) of cholesterol only. Platelet size and protein content decreased significantly, but the whole blood platelet count did not change. The platelets became enriched in cholesterol, as indicated by a significant increase in the cholesterol:phospholipid molar (C/P) ratio. Specific responses of washed platelets stimulated with various agonists were studied to determine the effects of hypercholesterolemia on the various pathways of platelet aggregation in the absence of plasma components. In platelets from hypercholesterolemic rabbits compared with controls: aggregation induced by ADP was not altered; collagen-induced responses (aggregation, secretion of [14C]serotonin from prelabelled platelets, thromboxane A2 (TXA2) formation, mobilization of [3H]arachidonate from prelabelled platelets) were enhanced; with aspirin-treated platelets, aggregation induced by the TXA2 mimetic U46619 was enhanced: and thrombin-induced responses of both untreated platelets (aggregation, secretion of granule contents, TXA2 formation) and aspirin-treated platelets (aggregation) were enhanced. Thus, platelets from cholesterol-fed rabbits not only form more TXA2, but they aggregate more extensively when stimulated by its mimetic. In addition, it has not been previously recognized that these platelets are also hypersensitive to thrombin-induced aggregation that is independent of TXA2.

Adenosine Diphosphate↗

PubMed1991

Rapid and selective measurement of platelet-activating factor using a quantitative bioassay of platelet aggregation.

A bioassay for the measurement of platelet-activating factor (PAF) based on the quantification of platelet aggregation was developed. The method used a platelet analyzer in conjunction with a multiwelled micromixing device and whole blood collected from male New Zealand White rabbits. This bioassay can be nonselective and used to quantitate platelet aggregation induced by any activator. The EC50 for platelet-activating factor, arachidonic acid, and adenosine diphosphate were 0.0232, 55, and 10 microM, and at these concentrations the half maximal aggregation response occurred at 7.5, 10.0, and 12.5 min, respectively. The bioassay was capable of the sensitive quantification of platelet aggregation in small volumes (50 microL) of citrated rabbit whole blood, using a short (i.e., 15-min) incubation period. This enabled multiple bioassays to be performed without the need for a large volume of whole blood to be collected from the rabbit. Selective measurement of platelet-activating factor was achieved by adding inhibitors of arachidonic acid- and adenosine diphosphate-dependent pathways of platelet activation, that is, acetylsalicyclic acid and phosphoenolpyruvate/pyruvate kinase, respectively, to the citrated rabbit whole blood immediately before bioassay. These inhibited arachidonic acid- and adenosine diphosphate-induced platelet aggregation, but had no effect on platelet aggregation induced by platelet-activating factor. Platelet-activating factor was selectively inhibited by its receptor antagonist BN 52021. This method for measuring platelet-activating factor was reproducible; at the EC50, the inter- and intrabioassay coefficients of variation were within acceptable limits at 13.17% and 9.75%, respectively.

Animals↗

PubMed1994

Activation of phospholipase D in human platelets by collagen and thrombin and its relationship to platelet aggregation.

Stimulation of phospholipase D after activation of cell surface receptors has been reported in many cell types. We have investigated the mechanism of activation of this enzyme by collagen in the human platelet by assaying the release of [3H]methylcholine from [3H]methylphosphatidylcholine. Results from these studies suggest that phospholipase D activity is regulated by reversible phosphorylation. Phospholipase D activity was stimulated when platelet-rich plasma was preincubated with collagen and was not inhibited by aspirin. Among various aggregating agents tested, collagen and thrombin but not ADP activated phospholipase D activity (2- to 3-fold). The addition of sphingosine inhibited phospholipase D activity. Preincubation of platelet-rich plasma with sphingosine inhibited collagen- and thrombin-induced platelet aggregation and the release of ATP. The inhibitory effect of sphingosine on collagen- and thrombin- induced platelet aggregation and release of ATP was dose-dependent. The functional significance of phospholipase D activation was also tested by examining the effect of the product, phosphatidic acid, on collagen-induced platelet aggregation and release of ATP. Platelet shape change and the reversibility of platelet aggregation resulted by the addition of phosphatidic acid to platelet-rich plasma. Furthermore, the simultaneous addition of phosphatidic acid and collagen shortened the latency period but had no effect on platelet aggregation. Two platelet proteins (47 kDa and 22 kDa) increased in phosphorylation after the addition of 1 microM phosphatidic acid which did not cause platelet aggregation. These results suggest that collagen stimulates phospholipase D activity which plays a secondary role in platelet aggregation and the release reaction.

Adenosine Triphosphate↗

PubMed1999

Platelet aggregatory response to platelet activating factor (PAF), ex vivo, and PAF-acetylhydrolase activity in patients with unstable angina: effect of c7E3 Fab (abciximab) therapy.

OBJECTIVE: Platelet activation and aggregation is a dominant feature in the pathophysiology of unstable angina. The final step of platelet aggregation is mediated through the platelet integrin glycoprotein IIb/IIIa (GP IIb/IIIa), while abciximab (ReoPro) is one of the most potent inhibitors of this receptor. Platelet-activating factor (PAF) is a potent platelet agonist which is degraded and inactivated by PAF-acetylhydrolase (PAF-AH). The plasma form of PAF-AH is associated with lipoproteins. We studied the platelet response to the aggregatory effect of PAF, ex vivo, in relation to the plasma PAF-AH activity in 32 patients with unstable angina, as well as the effect of abciximab therapy on the above parameters. METHODS: Thirty two patients with unstable angina and 25 sex- and age-matched healthy controls participated in the study. On the day of admission (day 1) 17 patients received a bolus of abciximab (0.25 mg/kg) followed by a 12-h infusion (10 micrograms/min). Platelet aggregation to both PAF and ADP, in platelet rich plasma, was successively studied in both patients receiving abciximab or remaining untreated. The plasma and HDL-associated PAF-AH activity was also determined at the same times. RESULTS: In the untreated patients, the PAF EC50 values were significantly lower on the day of admission, whereas the maximal percentage of aggregation was significantly higher compared to controls (p < 0.01 for both comparisons). Similar behaviour of the platelets was observed in the aggregatory effect of ADP. This aggregatory response was not significantly altered 4 days, 7 days or 1 month afterwards. In the 17 patients who received abciximab, platelet aggregation to both PAF and ADP was inhibited by 90 +/- 5 and 96 +/- 3%, respectively, 1 h after bolus. At 2 and 3 days after treatment, platelet aggregation to both agonists was significantly recovered being similar to controls. However, it was fully restored 6 days after bolus, still being significantly higher compared to controls (p < 0.01 for PAF and p < 0.003 for ADP). The total plasma PAF-AH activity in both patient groups was not different from that of controls, whereas the HDL-associated PAF-AH activity was significantly lower. The total plasma or HDL-associated enzyme activity was not altered at any time interval studied, and it was not influenced by abciximab. CONCLUSIONS: The increased aggregatory response of platelets to PAF and the low plasma levels of HDL-cholesterol and HDL-associated PAF-AH activity in patients with unstable angina may contribute to the severe atherosclerosis and to acute thrombosis found in these patients. Abciximab therapy may protect platelets from PAF action in vivo the first days after drug administration, but it fails to permanently restore the enhanced aggregatory response observed.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗

PubMed1998

Alimentary lipemia enhances the membrane expression of platelet P-selectin without affecting other markers of platelet activation.

The present study was conducted to determine whether alimentary lipemia alters platelet activity in vivo. Normolipidemic volunteers were given a fatty meal and platelet function was assessed before, and 3 and 6 h after the meal. Platelet aggregability and secretion was determined using whole blood flow cytometry (expression of platelet P-selectin and fibrinogen binding), filtragometry ex vivo (reflecting platelet aggregability in vivo) and by measurements of platelet specific products in plasma (beta-thromboglobulin and platelet factor 4). Plasma triglycerides increased from 0.8 (0.6:1.1; median, 25th and 75th percentiles) to 1.7 (1.0:2.3) mmol/l at 3 h and returned to baseline after 6 h (P < 0.001, one-way ANOVA). Apo B-100 and apo B-48 were both markedly increased 3 h postprandially in the Sf 60-400 fraction (large VLDLs, P < 0.001 for both), whereas the Sf 20-60 (small VLDLs) and Sf 12-20 fractions (IDL) did not change. The platelet function assessments revealed that the percentage of platelets expressing P-selectin increased by 40% (5%; 64%) after 3 h and by 51% (- 7%; 85%) 6 h postprandially in unstimulated samples (P < 0.05 for both). In samples stimulated by ADP in vitro P-selectin expression increased by 45% (6%; 58%) after 3 h and by 30% (12%; 58%) (P<0.01 for both) after 6 h at 0.1 microM. Platelet P-selectin expression was less influenced at higher ADP concentrations. The plasma levels of beta-thromboglobulin (approximately 20 ng/ml) and platelet factor 4 (approximately 0.3 ng/ml) were not affected by the fat load. Flow cytometric analyses of fibrinogen binding and filtragometry measurements also failed to reveal any postprandial alterations. The present finding of enhanced platelet P-selectin expression suggests that platelets are mildly sensitized postprandially. Whether this is of importance for thrombus formation and atherosclerosis needs to be studied further.

Adult↗

PubMed1997

Signal transduction system in epinephrine stimulated platelets; comparison between epinephrine sensitive and insensitive platelets.

We recently reported the high prevalence of impaired platelet responsiveness only to epinephrine in healthy Japanese. This abnormality was associated with a 50% decrease in the number of alpha 2-adrenergic receptors. Platelets from non-responders (NR) do not undergo secondary platelet aggregation even after exposure to 100 microM epinephrine, but they can potentiate the effect of ADP to provoke platelet aggregation. To further define the nature of the defect and to delineate controversial steps of epinephrine stimulated signal transduction, a signaling pathway of epinephrine was investigated in platelets from NR and R(normal responder to epinephrine). In a unique particle counting apparatus, epinephrine initially triggered the formation of small platelet aggregates composing of 10-1000 cells from both R and NR, but the aggregates became larger (4600 > cells) only in platelets from R. Thus, platelets from NR lack the ability to form larger aggregates. A similar defect was reproduced by treating normal platelets with aspirin. In the presence of fibrinogen, platelets from NR lacked phospholipase A2 activation, determined by arachidonic acid liberation in the presence of inhibitors to cyclooxygenase and lipoxygenase. In the absence of fibrinogen, aggregation and phospholipase A2 activation were not evident in R and NR. The surface expression of GPIIb/IIIa was markedly decreased in platelets from NR after stimulation by epinephrine, in comparison with those from R. The resting level and epinephrine stimulated increase in cAMP were not significantly different between NR and R. Incubating R platelets with a half saturating dose of yohimbine rendered them insensitive to epinephrine. These results indicated that the impaired platelet aggregation induced by epinephrine was due to the impaired surface exposure of glycoproteins GPIIbIIIa integral to the activation of phospholipase A2, which requires the full and normal occupancy of the alpha 2-adrenergic receptor by epinephrine.

Adenosine Triphosphate↗

PubMed1996

Flow cytometric detection of platelet-reactive antibodies and application in platelet crossmatching.

BACKGROUND: Alloimmunization against HLA or platelet antigens can cause refractoriness to platelet transfusions in multiply transfused patients. Crossmatching of platelet concentrates is effective in overcoming this problem. STUDY DESIGN AND METHODS: A flow cytometric assay was used for simultaneous detection of lymphocyte-reactive and platelet-reactive antibodies in a single sample using fluorescein isothiocyanate-labeled anti-IgG. This assay was compared with the monoclonal antibody-specific immobilization of platelet antigens (MAIPA) assay in selected sera containing HLA and platelet antibodies. In a further study, this assay was compared with lymphocytotoxicity test results from thrombocytopenic patients, for whom platelet concentrates were ordered. The results of both assays were then correlated with the 1-hour corrected count increment, with a corrected count increment greater then 7500 considered as an adequate transfusion response. RESULTS: The results of the MAIPA and flow cytometric assay in detecting platelet-reactive antibodies correlated well (p<0.0001, r = 0.84). The sensitivity and specificity of the flow cytometric assay in detecting platelet-reactive antibodies were 94.7 and 96.3 percent, when the MAIPA assay was taken as a reference. In unselected sera from patients, the sensitivity and specificity of the flow cytometric assays were, respectively, 72.7 and 91.7 percent in detecting lymphocyte-reactive antibodies and 70.6 and 77.7 percent in detecting platelet-reactive antibodies, when the lymphocytotoxicity test was used as a reference. With regard to an adequate transfusion response, the sensitivities and efficiencies were 20.0 and 82.1 percent, 33.3 and 84.3 percent, and 70.0 and 88.6 percent for the lymphocytotoxicity test and the lymphocyte-reactive and platelet-reactive flow cytometric assays, respectively. CONCLUSION: Flow cytometric crossmatching appears to be an effective method of detecting platelet-reactive antibodies that may affect the success of platelet transfusions. This procedure is well-suited for routine conditions and can be performed within 2 hours.

Antibodies, Monoclonal↗

PubMed2002

Growth factor levels in platelet-rich plasma and correlations with donor age, sex, and platelet count.

INTRODUCTION: Platelet-rich plasma contains autologous thrombocyte growth factors and might be promising for acceleration of dentoalveolar bone regeneration. In this study, it was analysed for platelet counts and growth factor concentrations. MATERIAL AND METHOD: Platelet-rich plasma was isolated by discontinuous cell separation from 158 healthy men and 55 women aged 17-62 years. One hundred and fifteen specimens (stratified for age and gender of the donor) were analysed for growth factor concentrations and platelet count. RESULTS: The platelet count in platelet-rich plasma (1,407,640+/-320,100/microl) was 5 times higher than in donor blood (266,040+/-60,530/microl). Platelet-derived growth factor AB (117+/-63 ng/ml), transforming growth factor (TGF) beta -1 (169+/-84 ng/ml), and insulin-like growth factor (IGF) I (84+/-23 ng/ml) were found in large amounts, while platelet-derived growth factor (PDGF) BB (10+/-8 ng/ml) and transforming growth factor beta -2 (0.4+/-0.3 ng/ml) were found in small amounts only. The growth factor content was not well correlated with the platelet count in whole blood nor with the platelet-rich plasma (r(p)=0.35). No influence of gender or age on platelet count or growth factor concentrations was discovered (except IGF-I). CONCLUSIONS: While there was substantial variation in the growth factor content of platelet-rich plasma, the factors influencing this are still worthy of further investigation. Furthermore, a technique whereby the growth factor content could be rapidly assessed in platelet-rich plasma may be of therapeutic benefit.

Adolescent↗

PubMed1996

Platelet-associated factor XIII in platelet activation, adhesion, and clot stabilization.

Platelet-associated factor XIII provides a means by which to promote clot stabilization and platelet interaction with proteins of the coagulation and fibrinolytic pathways. In addition to its intracellular role within the platelet cytoplasm, activated factor XIII will bind to the surface of activated platelets. These platelets then participate in cell-cell or cell-clot interactions, thereby increasing the local concentration of factor XIIIa. The platelet-associated factor XIIIa may increase the amount of crosslinking in a fibrin clot, thereby contributing to the aging of the clot and the reduction in the degree of platelet binding. Clot resistance to fibrinolysis is enhanced by platelet factor XIIIa-mediated crosslinking of alpha 2-antiplasmin to fibrin. The binding of factor XIIIa to the platelet surface requires the activation of the platelet fibrinogen receptor, glycoprotein IIb-IIIa. Thus, platelet-associated factor XIIIa may be used as a marker of in vivo platelet activation. Since half of the factor XIII present in blood is provided by the platelets, it is not surprising that this form of factor XIII plays an important role in hemostasis.

Blood Coagulation↗

PubMed2002

Comparative efficacy of fibrinogen and platelet supplementation on the in vitro reversibility of competitive glycoprotein IIb/IIIa receptor-directed platelet inhibition.

BACKGROUND: Platelet surface glycoprotein IIb/IIIa (alphaIIb/beta3) receptor inhibition, with prevention of fibrinogen binding and platelet aggregation, concomitantly attenuates arterial thrombotic capacity and impairs protective hemostasis, 2 divergent platelet-dependent processes. PURPOSE: Because the currently available, Food and Drug Administration-approved small molecule glycoprotein IIb/IIIa receptor antagonists are considered "competitive" inhibitors and there is limited information on the reversibility of platelet inhibition with fibrinogen or platelet supplementation, the following series of in vitro experiments were performed. METHODS AND RESULTS: Washed platelets from 24 healthy volunteers were suspended in tyrodes buffer and incubated with achievable (in vivo) concentrations of either tirofiban or eptifibatide before activation with thrombin receptor agonist peptide (15 micromol/L). Platelet aggregation was inhibited by 40% to 50%, but reversal was achieved with fibrinogen supplementation in a concentration-dependent manner. In a separate series of in vitro experiments, platelet inhibition exceeding 90% was established with tirofiban (average concentration 9.28 microg/L) and eptifibatide (average concentration 95.4 microg/L). Recovery of platelet aggregation to at least 50% was achieved after the addition of fibrinogen (0.76-0.80 g/L), platelets (2.4 x 10(11)/L), or their combination. There was an inverse relationship between plasma baseline fibrinogen and the amount of supplemental fibrinogen needed to restore platelet aggregability (r = -0.60; P <.01). CONCLUSION: The reversibility of glycoprotein IIb/IIIa-directed platelet inhibition is influenced by cell surface receptor availability and intrinsic pharmacodynamic mechanism of action. Fibrinogen supplementation with fresh frozen plasma or cryoprecipitate either alone or in combination with platelet transfusion represents an important and readily available treatment consideration for restoring hemostatic potential and managing major hemorrhagic complications associated with the administration of small-molecule, competitive glycoprotein IIb/IIIa receptor antagonists.

Adult↗

PubMed2003

Unique in vivo modifications of coagulation factor V produce a physically and functionally distinct platelet-derived cofactor: characterization of purified platelet-derived factor V/Va.

Platelet- and plasma-derived factor Va (FVa) serve essential cofactor roles in prothrombinase-catalyzed thrombin generation. Platelet-derived FV/Va, purified from Triton X-100 platelet lysates was composed of a mixture of polypeptides ranging from approximately 40 to 330 kDa, mimicking those visualized by Western blotting of platelet lysates and releasates with anti-FV antibodies. The purified, platelet-derived protein expressed significant cofactor activity such that thrombin activation led to only a 2-3-fold increase in cofactor activity yet expression of a specific activity identical to that of purified, plasma-derived FVa. Physical and functional differences between the two cofactors were identified. Purified, platelet-derived FVa was 2-3-fold more resistant to activated protein C-catalyzed inactivation than purified plasma-derived FVa on the thrombin-activated platelet surface. The heavy chain subunit of purified, platelet-derived FVa contained only a fraction ( approximately 10-15%) of the intrinsic phosphoserine present in the plasma-derived FVa heavy chain and was resistant to phosphorylation at Ser(692) catalyzed by either casein kinase II or thrombin-activated platelets. MALDI-TOF mass spectrometric analyses of tryptic digests of platelet-derived FV peptides detected an intact heavy chain uniquely modified on Thr(402) with an N-acetylglucosamine or N-acetylgalactosamine, whereas Ser(692) remained unmodified. N-terminal sequencing and MALDI-TOF analyses of platelet-derived FV/Va peptides identified the presence of a full-length heavy chain subunit, as well as a light chain subunit formed by cleavage at Tyr(1543) rather than Arg(1545) accounting for the intrinsic levels of cofactor activity exhibited by native platelet-derived FVa. These collective data are the first to demonstrate physical differences between the two FV cofactor pools and support the hypothesis that, subsequent to its endocytosis by megakaryocytes, FV is modified to yield a platelet-derived cofactor distinct from its plasma counterpart.

Blood Coagulation↗

PubMed1990

Effects of streptokinase, urokinase, and recombinant tissue plasminogen activator on platelet aggregability and stability of platelet aggregates.

STUDY OBJECTIVE: The aim of the study was to evaluate the effects of streptokinase, urokinase and recombinant tissue plasminogen activator (TPA) on platelet aggregability and metabolism and the stability of preformed platelet aggregates. DESIGN: The experiments (n = 15 for each condition) were performed on citrated plasma or on platelet suspensions in phosphate buffered saline, both with a standardised platelet count of 250 x 10(9).litre-1. SUBJECTS: were healthy volunteers. MEASUREMENTS AND MAIN RESULTS: With both ADP (1 mumol.litre-1) and collagen (1 mg.litre-1) as aggregating agents, streptokinase at greater than or equal to 10(5) units.litre-1 led to reduction in the rate of platelet aggregation. With collagen and in most instances with ADP, this was associated with a decreased extent of aggregation, though in five out of 30 cases with ADP as aggregating agent, a conversion from reversible to irreversible aggregation occurred with streptokinase. Urokinase inhibited platelet aggregation at greater than or equal to 3 x 10(5) units.litre-1 with both aggregating agents. TPA inhibited aggregation at greater than or equal to 1 mg.litre-1 with ADP and at greater than or equal to 3.3 mg.litre-1 with collagen as aggregating agent. The inhibitory effect was still present when the platelets were suspended in saline. Platelet synthesis of thromboxane on stimulation with collagen, and of c-AMP on stimulation with prostaglandin E1, was markedly reduced by either agent. The stability of platelet aggregates, as assessed photometrically during a 90 min exposure to stirring stress, increased when streptokinase or urokinase was added to platelet rich plasma, but remained uninfluenced with TPA. CONCLUSIONS: Urokinase and TPA inhibited platelet aggregability uniformly and in a dose dependent manner. Streptokinase inhibited platelet aggregation in most instances, but led to a stimulation of aggregation in a minority of cases. These effects of the thrombolytic agents on platelets might have an influence on the occurrence of bleeding and of reocclusion after thrombolytic therapy.

Adenosine Diphosphate↗

PubMed1975

Hereditary giant platelet syndrome. Absence of collagen-induced coagulant activity and deficiency of factor-XI binding to platelets.

The platelets from two related patients with the hereditary giant platelet syndrome were examined. They were larger than normal but otherwise ultrastructurally normal; they contained increased storage pools of adenine nucleotides and heparin-neutralizing activity and took up serotonin at an increased rate. They aggregated normally with ADP and collagen but failed to aggregate with bovine factor VIII and Ristocetin. Some change in shape occurred with ADP, and the reduction in adenylate energy change after addition of ADP to platelet-rich plasma was smaller than normal. Platelet coagulant activities including contact product forming activity, intrinsic factor-Xa forming activity and platelet factor 3 activity were normal or increased, but collagen-induced coagulant activity was absent whether tested in washed platelet suspensions or platelet-rich plasma. Platelet washing experiments showed decreased binding of factors V and VIII to hereditary giant platelets and no detectable factor XI in washed platelet suspensions. It is concluded that (1) the hereditary giant platelets studied lacked a binding mechanism for factors, V, VIII and XI; (2) the normal development of collagen-induced coagulant activity apparently depends upon the binding of factor XI to the platelet membrane; and (3) the defective prothrombin consumption observed in these patients may have resulted from the failure of their platelets to bind factor XI.

Adenine Nucleotides↗