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PubMed1990

Acid treatment of platelets as a simple procedure for distinguishing platelet-specific antibodies from anti-HLA antibodies: comparison with chloroquine treatment.

The identification of antibodies to platelet-specific antigens is important for correctly diagnosing neonatal alloimmune thrombocytopenia, posttransfusion purpura and refractoriness due to platelet-specific antibodies. However, the serologic identification of these platelet-specific antibodies is complicated by the presence of anti-HLA antibodies. We examined and compared the diagnostic usefulness of acid-treated and chloroquine-treated platelets for the discrimination of platelet-specific antibodies from anti-HLA antibodies. The viability of acid-treated platelets is 83.4%, which is better than that of chloroquine-treated platelets (52.6%). The antigenicity of HLA class I antigens of acid-treated platelets was significantly reduced compared with that of PBS- or chloroquine-treated platelets. On the other hand, platelet surface glycoprotein Ib and glycoprotein IIb/IIIa, and platelet-specific antigens were stable following acid or chloroquine treatment. Chloroquine-treated platelets were not suitable targets for analysis by immunofluorescence flow cytometry because of nonspecific fluorescence derived from platelet damage. We conclude that acid-treated platelets are more suitable targets than chloroquine-treated platelets for screening for platelet-specific antibodies and also for analyses of the specificity of platelet-specific antibodies.

Antibody Specificity↗
PubMed1997

Platelet integrin alpha IIb beta 3 (GPIIb-IIIa) is not implicated in the binding of LDL to intact resting platelets.

It has been suggested that the fibrinogen receptor (glycoprotein [GP] IIb-IIIa or platelet integrin alpha IIb beta 3) could be the binding site for low-density lipoprotein (LDL); however, recent data do not support this. Furthermore, GPIIb and not the GPIIb-IIIa complex is the main binding protein for lipoprotein(a) [Lp(a)]. In the present study, we have investigated the interaction between Lp(a) particles and platelet LDL binding sites and whether platelet integrin alpha IIb beta 3 is implicated. Displacement experiments showed that 125I-LDL binding to intact resting platelets was inhibited with the same apparent affinity by both unlabeled LDL and apolipoprotein(a)-free lipoprotein particles [Lp(a)-, an LDL-like particle prepared from Lp(a)]. Hill coefficients for displacement curves suggested that a single set of binding sites was involved. In contrast, both native and oxidized Lp(a) particles were unable to inhibit platelet LDL binding. Furthermore, platelets bound 125I-Lp(a)- particles to a class of saturable binding sites numbering approximately 1958 +/- 235 binding sites per platelet with a dissociation constant (Kd) of 48.3 +/- 12 x 10(-9) mol/L. These values were similar to those obtained for LDL. In contrast to Lp(a), evidence indicates that platelet integrin alpha IIb beta 3 was not involved in the interaction of LDL and intact resting platelets. First, specific ligands for platelet integrin alpha IIb beta 3, such as fibrinogen, vitronectin, and fibronectin, were unable to inhibit the binding of LDL to intact resting platelets. Second, similar LDL binding characteristics (Kd and Bmax values) were found in platelets from control subjects and patients with type I and type II Glanzmann's thrombasthenia, characterized by total and partial lack of GPIIb-IIIa and fibrinogen, respectively. Third, polyclonal antibodies against the GPIIb-IIIa complex (edu-3 and 5B12), human antiserums against platelet alloantigens (anti-Baka/B and anti-PLA1/2), anti-integrin subunits (anti-alpha v and anti-beta 3), and a wide panel of monoclonal antibodies (mAbs) against well-known epitopes of GPIIb (M3, M4, M5, M6, and M95-2b) and GPIIIa (P23-7, P33, P37, P40, and P97) did not affect platelet LDL binding. Finally, in contrast to the proaggregatory effect of native and oxidized LDL, both native and oxidized Lp(a) particles caused a significant dose-dependent decrease of collagen-induced platelet aggregation. In conclusion, we demonstrate that neither the GPIIb-IIIa complex nor GPIIb and GPIIIa individually are membrane binding proteins for LDL on intact resting platelets. Lp(a) particles do not interact with platelet LDL binding sites, and their biological response is clearly different from that of LDL.

Binding Sites↗
PubMed1998

Neutrophil-platelet adhesion: relative roles of platelet P-selectin and neutrophil beta2 (DC18) integrins.

Neutrophils and platelets interact both physically and metabolically during inflammation and thrombosis, but the mechanisms responsible for their adhesion remain incompletely understood. Neutrophil-platelet adhesion was measured after specific stimulation of neutrophils, platelets, or both and quantified by flow cytometry. Specific stimulation of either the neutrophil or the platelet led to a marked increase in the percentage of neutrophils that bound platelets, although platelet stimulation led to a large increase and neutrophil stimulation to only a small increase in the number of platelets per neutrophil. Stimulation of both cells further increased the number of neutrophil-platelet adhesive events and led to large numbers of platelets binding to each neutrophil. Confirming previous observations, blocking antibodies to platelet P-selectin (CD62P) partially inhibited adhesion. However, blockade of the neutrophil beta2 integrin CD11b/CD18 also inhibited the percentage of neutrophils that bound platelets. Combining P-selectin and CD11b/18 blockade further inhibited the stimulated increase in the percentage of neutrophils binding platelets and the increased number of platelets per neutrophil. Both cell adhesion molecules were active even when only a single cell type was primarily activated, supporting physiologically important transcellular activation. These data suggest that: (1) neutrophil-platelet adhesion can be initiated by specific activation of either the neutrophil or the platelet and that specific activation of either cell type leads to distinct patterns of adhesion, and (2) neutrophil-platelet adhesion uses both platelet P-selectin and the neutrophil beta2 integrin CD11b/CD18 when the cells are primarily or secondarily activated.

Antibodies↗
PubMed2001

Alterations of platelet aggregation kinetics with ultraviolet laser emission: the "stunned platelet" phenomenon.

Platelets, a major constituent of thrombus, play a crucial role in the pathogenesis of acute ischemic coronary syndromes. The effect of ultraviolet laser emission on platelets within thrombi is unknown. The effects of increasing levels of laser energy on platelets in whole blood were investigated. Blood samples were obtained by aseptic venipuncture and anticoagulated with 3.8% sodium citrate. Samples were exposed to increased levels (0, 30, 45, 60 mJ/mm2; 25 Hz) of ultraviolet excimer laser fluence (308 nm wave-length) and then tested for ADP and collagen induced platelet aggregation, platelet concentration, and for platelet contractile force (PCF) development. Scanning electron microscopy was used to detect laser induced morphologic changes of platelets and by flow cytometric analysis to detect changes in expression of platelet surface antigens p-selectin (CD 62) and glycoprotein IIb/IIIa (CD 43). Exposure to excimer laser energy produced dose dependent suppression of platelet aggregation and force development ("stunned platelets"). ADP aggregation decreased from 8.0+/-1.1 Ohms (mean+/-SEM) to 3.7+/-0.8 Ohms (p<0.001) to 2.7+/-0.6 Ohms (p <0.001) and to 1.8+/-0.5 Ohms (p <0.001) as the laser energy increased from 0 to 30 to 45 to 60 mJ/mm2, respectively. Collagen induced aggregation decreased from 21.4+/-1.4 Ohms to 15.7+/-1.2 Ohms (p <0.001) to 11.7+/-1.1 Ohms (p <0.001) and to 9.9+/-1.0 Ohms (p <0.001), in response to the same incremental range of laser energy. Platelet contractile forces declined from 34,500+/-3700 to 27.800+/-2700 dynes as laser energy increased from 0 to 60 mJ/mm2 (p <0.03). Platelet concentration did not change with increasing laser energy. The expression of platelet surface antigen p-selectin (CD 62) remained stable through increasing levels of laser energy exposures while the percentage of CD 43 positive platelets significantly increased with exposure to laser energy, yet the level of expression did not exceed 0.5% of cells. Thus, aggregation kinetics are altered in platelets exposed to ultraviolet laser energy as manifested by decreased platelet aggregation and reduction in platelet force development capability. The response is dose dependent and most pronounced at higher energy levels such as 60 mJ/mm2.

Adenosine Diphosphate↗
PubMed1991

Characterization of the binding of thrombospondin to human platelets and its association with the platelet cytoskeleton.

To characterize the interaction between thrombospondin and human platelets, thrombospondin was purified from the supernatant of thrombin-activated human platelets, labeled with iodine 125, and allowed to interact with the washed platelets. With concentrations of 10 to 50 micrograms/ml, only minute amounts of 125I-labeled thrombospondin bound to resting platelets or to platelets activated by adenosine diphosphate. In contrast, when platelets were stimulated with thrombin, binding increased fivefold to sixfold in a time-dependent and 125I-labeled thrombospondin concentration-dependent manner. Binding of 125I-labeled thrombospondin to thrombin-activated platelets required the presence of divalent cations, proceeded concomitantly with platelet release, and at a concentration of 1 nmol/L thrombin, reached a maximum of 2200 +/- 260 molecules of 125I-labeled thrombospondin bound per platelet. After its binding to platelets, 125I-labeled thrombospondin was not internalized, because up to 85% of the 125I-labeled thrombospondin was dissociated from the cell surface by adding ethylenediaminetetraacetic acid. Using various experimental approaches, including studies with severe type I thrombasthenic platelets, we further demonstrated that the interaction of 125I-labeled thrombospondin with thrombin-stimulated platelets occurred as a fibrinogen- and fibrin-independent process, and that the glycoprotein IIb-IIIa complex did not function as a physiologic plasma membrane receptor for 125I-labeled thrombospondin. Last, about 60% of the 125I-labeled thrombospondin molecules bound to the platelet surface were found to be associated with the platelet cytoskeleton recovered from platelets solubilized with Triton X-100. On Western blot analysis, this cytoskeletal fraction lacked detectable glycoprotein IV, the putative platelet receptor for thrombospondin. These results suggest that on the surface of thrombin-activated platelets, a fraction of 125I-labeled thrombospondin does not associate with glycoprotein IV but instead with other plasma membrane components that have yet to be identified.

Blood Platelets↗
PubMed1989

Thrombin-induced increase in surface expression of epitopes on platelet membrane glycoprotein IIb/IIIa complex and GMP-140 is a function of platelet age.

Platelets are heterogeneous in the content of membrane glycoprotein (GP)IIb/IIIa complex. To determine whether this heterogeneity is related to changes associated with platelet aging in the circulation, newly released platelets, obtained during recovery from nonimmune-mediated acute experimental thrombocytopenia in baboons, were studied. Monoclonal antibody (MoAb) binding to epitopes expressed on GPIIb/IIIa complex (LJ-CP8), GMP-140 (S12), and GPIa/IIa (12F1) was measured on control platelets (comprising platelets with a normal age distribution; mean age 60 to 72 hours) and newly formed platelets (mean age 12 hours), both in the resting state and after thrombin stimulation. Whereas LJ-CP8 binding to resting control platelets increased by 34% upon stimulation by gamma-thrombin from 30,885 +/- 1,171 to 41,458 +/- 1,311 molecules/platelet at saturating concentrations of antibody, LJ-CP8 binding to resting young platelets did not increase significantly upon thrombin stimulation (31,878 +/- 3,330 and 33,791 +/- 3,486 molecules/platelet, respectively). Similarly, binding of antibody S12 in response to maximal thrombin stimulation was reduced by 42% from 10,246 +/- 834 molecules/platelet at saturating concentrations of S12 for control platelets to 5,971 +/- 665 molecules/platelet for young platelets (P = .001). S12 binding to unstimulated platelets was less than 10% of the binding observed after thrombin stimulation at all concentrations of S12 for both control and young platelets. However, maximal binding of antibody 12F1 to resting control platelets did not differ significantly from that observed with resting young platelets (2,926 +/- 167 and 2,857 +/- 208 molecules/platelet, respectively), and 12F1 binding was unchanged after thrombin stimulation for both control and young platelets. We conclude that the thrombin-induced increase in the expression of epitopes on platelet membrane GPIIb/IIIa complex and GMP-140 is a function of platelet age.

Animals↗
PubMed1985

Platelet kinetics in patients with bone marrow hypoplasia: evidence for a fixed platelet requirement.

We have studied 16 normal subjects and 27 patients with stable, untreated thrombocytopenia secondary to bone marrow failure and platelet counts ranging from 12,000 to 70,000/microL. Autologous platelets were labeled with 51Cr for measurement of mean platelet life span in the normal subjects and in 20 patients. Labeled donor cells were used in the remaining subjects. Platelet survival, as determined with both autologous and homologous platelets, correlated directly with platelet count in the thrombocytopenic patients. Platelet life span was only modestly reduced in patients having counts in the range of 50,000 to 100,000/microL (7.0 +/- 1.5 days v 9.6 +/- 0.6; P less than .01) but was markedly reduced when the count fell below 50,000/microL (5.1 +/- 1.9 days, P less than .001). The recovery of donor platelets in severely thrombocytopenic recipients (60% +/- 15%) was equivalent to control values (66% +/- 8%; P greater than .2). The recovery of autologous platelets was normal when the platelet count exceeded 50,000/microL (74% +/- 15%) but was reduced in patients with lower counts (50% +/- 22%; P less than .01). All patient and normal data were well correlated by a model predicting a maximum platelet life span of 10 1/2 days and a fixed requirement for 7,100 platelets per microliter of blood per day, or about 18% of the normal rate of platelet turnover, which averaged 41,200 platelets per microliter per day. We conclude that although relatively few platelets are used to support vascular integrity, this requirement is reflected by a reduced platelet life span in marrow hypoplasia and may contribute to the shortening of platelet survival observed in other thrombocytopenias.

Adolescent↗
PubMed1984

Platelet size and age determine platelet function independently.

This study was undertaken to examine the interaction of platelet size and age in determining in vitro platelet function. Baboon megakaryocytes were labeled in vivo by the injection of 75Se-methionine. Blood was collected when the label was predominantly associated with younger platelets (day 2) and with older platelets (day 9). Size-dependent platelet subpopulations were prepared on both days by counterflow centrifugation. The reactivity of each platelet subpopulation was determined on both days by measuring thrombin-induced aggregation. Platelets were fixed after partial aggregation had occurred by the addition of EDTA/formalin. After removal of the aggregated platelets by differential centrifugation, the supernatant medium was assayed for remaining platelets and 75Se radioactivity. Comparing day 2 and day 9, no significant difference was seen in the rate of aggregation of a given subpopulation. However, aggregation was more rapid in the larger platelet fractions than in the smaller ones on both days. A greater percentage of the 75Se radioactivity appeared in the platelet aggregates on day 2 than on day 9. This effect was independent of platelet size, as it occurred to a similar extent in the unfractionated platelets and in each of the size-dependent platelet subpopulations. The data indicate that young platelets are more active than older platelets. This study demonstrates that size and age are both determinants of platelet function, but by independent mechanisms.

Animals↗
PubMed1981

Detection of platelet alloimmunity with a platelet-associated IgG assay.

A quantitative immunofluorescence PA-IgG assay was used to detect alloimmunity to platelets. The assay identified serum alloantibodies in 10 out of 14 multitransfused patients and for two of three infants with neonatal thrombocytopenia. The correct separation of all multitransfused patients into alloimmune and nonalloimmune groups by the PA-IgG assay was substantiated with chromium-51--labeled platelet survival studies. The allogeneic nature of the serum antibodies was demonstrated by progressive absorption of the antibody with increasing numbers of allogeneic platelets but not with autologous platelets. The sensitivity of the PA-IgG assay for detection of serum alloantibodies was superior to that of platelet aggregation, platelet serotonin release, and lymphocytotoxicity testing. In dilution experiments with alloimmune serum, elevated levels of serum PA-IgG could still be detected on donor platelets when platelet aggregation and serotonin release tests became negative. Platelet survival studies with selected platelets performed in the 10 alloimmunized, multitransfused patients confirmed the results of the PA-IgG assays, predicting alloimmunity to the donor platelets. In contrast, platelet aggregation, platelet serotonin release, and lymphocytotoxicity testing indicated alloimmunity for 50% or less of the patients. Reduced platelet survival times were also seen with HLA A- and HLA B-matched donor platelets when donor-recipient incompatibility was demonstrated by the PA-IgG assay. Thus the PA-IgG assay provides a sensitive method to detect serum platelet alloantibodies and may offer a technique in platelet crossmatching.

Absorption↗
PubMed1995

Staphylococcus aureus induces platelet aggregation via a fibrinogen-dependent mechanism which is independent of principal platelet glycoprotein IIb/IIIa fibrinogen-binding domains.

Platelet aggregation by bacteria is felt to play an important role in the pathogenesis of infective endocarditis. However, the mechanisms involved in bacterium-induced platelet aggregation are not well-defined. In the present study, we examined the mechanisms by which Staphylococcus aureus causes rabbit platelet aggregation in vitro. In normal plasma, the kinetics of S. aureus-induced platelet aggregation were rapid and biphasic. The onset and magnitude of aggregation phase 1 varied with the bacterium-platelet ratio, with maximal aggregation observed at a ratio of 5:1. The onset of aggregation phase 2 was delayed in the presence of apyrase (an ADP hydrolase), suggesting that this later aggregation phase may be triggered by secreted ADP. The onset of aggregation phase 2 was delayed in the presence of prostaglandin I2-treated platelets, and this phase was absent when paraformaldehyde-fixed platelets were used, implicating platelet activation in this process. Platelet aggregation phase 2 was dependent on S. aureus viability and an intact bacterial cell wall, and it was mitigated by antibody directed against staphylococcal clumping factor (a fibrinogen-binding protein) and by the cyclooxygenase inhibitor indomethacin. Similarly, aggregation phase 2 was either delayed or absent in three distinct transposon-induced S. aureus mutants with reduced capacities to bind fibrinogen in vitro. In addition, a synthetic pentadecapeptide, corresponding to the staphylococcal binding domain in the C terminus of the fibrinogen delta-chain, blocked aggregation phase 2. However, phase 2 of aggregation was not inhibited by two synthetic peptides (alone or in combination) analogous to the two principal fibrinogen-binding domains on the platelet glycoprotein (GP) IIb/IIIa integrin receptor: (i) a recognition site on the IIIa molecule for the Arg-Gly-Asp (RGD) sequence of the fibrinogen alpha-chain and (ii) a recognition site on the IIb molecule for a dodecapeptide sequence of the fibrinogen delta-chain. This differs from ADP-induced platelet aggregation, which relies on an intact platelet GP IIb/IIIa receptor with an accessible RGD sequence and dodecapeptide recognition site for fibrinogen. Furthermore, a monoclonal antibody directed against the RGD recognition site on rabbit platelet GP IIb/IIIa receptors failed to inhibit rabbit platelet aggregation by S. aureus. Collectively, these data suggest that S. aureus-induced platelet aggregation requires bacterial binding to fibrinogen but is not principally dependent upon the two major fibrinogen-binding domains on the platelet GP IIb/IIIa integrin receptor, the RGD and dodecapeptide recognition sites.

Adenosine Triphosphate↗
PubMed1991

High molecular weight kininogen inhibits thrombin-induced platelet aggregation and cleavage of aggregin by inhibiting binding of thrombin to platelets.

In this study we show that high molecular weight kininogen (HK) inhibited alpha-thrombin-induced aggregation of human platelets in a dose-dependent manner with complete inhibition occurring at plasma concentration (0.67 mumol/L) of HK. HK (0.67 mumol/L) also completely inhibited thrombin-induced cleavage of aggregin (Mr = 100 Kd), a surface membrane protein that mediates adenosine diphosphate (ADP)-induced shape change, aggregation, and fibrinogen binding. The inhibition of HK was specific for alpha- and gamma-thrombin-induced platelet aggregation, because HK did not inhibit platelet aggregation induced by ADP, collagen, calcium ionophore (A23187), phorbol myristate acetate (PMA), PMA + A23187, or 9,11-methano derivative of prostaglandin H2 (U46619). These effects were explained by the ability of HK, at physiologic concentration, to completely inhibit binding of 125I-alpha-thrombin to washed platelets. As a result of this action of HK, this plasma protein also completely inhibited thrombin-induced secretion of adenosine triphosphate, blocked intracellular rise in Ca2+ in platelets exposed to alpha- and gamma-thrombin, inhibited thrombin-induced platelet shape change, and blocked the ability of thrombin to antagonize the increase in intracellular cyclic adenosine monophosphate (cAMP) levels induced by iloprost. Because elevation of cAMP is known to inhibit binding of thrombin to platelets, we established that HK did not increase the intracellular concentration of platelet cAMP. Finally, HK did not inhibit enzymatic activity of thrombin. To study the role of HK in the plasma environment, we used gamma-thrombin to avoid fibrin formation by alpha-thrombin. Platelet aggregation induced by gamma-thrombin was also inhibited by HK in a dose-dependent manner. The EC50 (concentration to produce 50% of the maximum rate of aggregation) of gamma-thrombin for washed platelets was 7 nmol/L and increased to 102 nmol/L when platelets were suspended in normal human plasma. The EC50 for platelet aggregation induced by alpha-thrombin in plasma deficient in total kininogen was 40 nmol/L. When supplemented with HK at plasma concentration (0.67 mumol/L), the EC50 increased to 90 nmol/L, a value similar to that for normal human plasma. These results indicate that (1) HK inhibits thrombin-induced platelet aggregation and cleavage of aggregin by inhibiting binding of thrombin to platelets; (2) HK is a specific inhibitor of platelet aggregation induced by alpha- and gamma-thrombin; and (3) HK plays a role in modulating platelet aggregation stimulated by alpha-thrombin in plasma.

Adenosine Triphosphate↗
PubMed2003

Triggers for prophylactic use of platelet transfusions and optimal platelet dosing in thrombocytopenic dogs and cats.

Prophylactic platelet transfusions are frequently given to human patients with hypoproliferative thrombocytopenia. For several decades, the most common transfusion trigger was 20,000/microL, but the trend is now to use 10,000/microL in the absence of other risk factors for bleeding. This trigger seems to reduce the number of transfusions without increasing the risk of severe bleeding. Most studies involved in establishing platelet transfusion policies have involved patients with acute leukemia, with fewer studies involving patients undergoing hematopoietic stem cell transplantation or aggressive chemotherapy for other cancers and patients with aplastic anemia. In the presence of other risk factors for spontaneous bleeding, 20,000/microL is still considered an appropriate trigger. The trigger for prophylactic transfusion before surgery has not undergone the same recent scrutiny as has the trigger for spontaneous bleeding. The recommendation remains to raise the platelet count to 50,000 to 100,000/microL if possible, although it is recognized that surgery and other invasive procedures have been performed at lower platelet counts without major bleeding. Prophylactic transfusion is not used in disorders of platelet consumption and destruction to prevent spontaneous bleeding but is used before surgery. Because of the comparative lack of experience with platelet transfusion in veterinary medicine, it is difficult to make generalizations for dogs and cats. Using the guidelines established for therapeutic and prophylactic transfusion of human patients is a reasonable starting point, however. A therapeutic transfusion policy is suggested in the veterinary setting provided that the patient can be closely observed for critical bleeding and a prompt transfusion can be given. This policy should ultimately reduce the overall number of platelet transfusions given to hospital patients. If an animal cannot be closely observed or the ability to transfuse on demand is limited, prophylactic transfusion is recommended. The triggers for initiating a platelet transfusion in dogs are extrapolated from human data; these values are lower by 50% for cats. Because of the imprecision of platelet counting at low values, platelet counts must always be interpreted in conjunction with clinical signs of hemorrhage. If platelet-rich plasma or platelet concentrate is available, a dose of 1 platelet unit per 10 kg is recommended, although resources may dictate a smaller dose. This will raise the recipient platelet count by a maximum of about 40,000/microL. Assuming a trigger of 10,000/microL, a transfusion will probably be required approximately every 3 days. It must be remembered that the frequency of platelet transfusions may be greater in the presence of factors accelerating platelet loss or destruction. If fresh whole blood is used, a rule of thumb is to transfuse 10 mL/kg, which will raise the recipient platelet count by a maximum of approximately 10,000/microL. Daily transfusions or transfusions every other day will probably be required.

Animals↗
PubMed1977

Platelet antithrombins: role of thrombin binding and the release of platelet fibrinogen.

The nature of platelet antithrombin was elucidated by comparison of thrombin binding and antithrombin activities of intact platelets and by purification of antithrombin from platelet lysates using glycerol osmotic lysis, ethanol precipitation and Sephadex gel filtration techniques. The major portion of the antithrombin and thrombin binding activity of intact platelets is lost after brief sonication. The antithrombin activity in destroyed platelets is found to be due to platelet fibrinogen. Treatment of platelets with PGE1 (100 microng/ml) markedly inhibits (greater than 80%) the release of platelet fibrinogen induced by thrombin. However, the PGE1 treatment produced slight (less than 30%) but significant decrease of antithrombin activity of intact platelets, whereas the binding of thrombin to platelets was not affected by PGE1 treatment. The amounts of thrombin bound to and inactivated by PGE1-treated platelets at the same cell concentration are identical. The above results suggest that platelets contain at least two antithrombin activities. One, which accounts for the major portion of platelet antithrombin is mediated by thrombin binding to platelets. The other, which attributes to a lesser extent to platelet antithrombin activity, is due to the release of platelet fibrinogen. Also, antithrombin is readily demonstrated in a plasma medium indicating physiological significance of platelet antithrombin.

Antithrombins↗
PubMed1988

Increased levels of platelet associated IgG in patients with thrombocytopenia are not confined to any particular size class of platelets.

Patients with immune thrombocytopenia have an increased percentage of microthrombocytes/platelet fragments and megathrombocytes. It has been suggested that increased levels of platelet associated IgG (PA-IgG) found in these patients might be related to the presence of this abnormal platelet size distribution. In this study we used flow cytometry to investigate the distribution of PA-IgG within a population of platelets and, in particular, we examined the relationship between platelet size and PA-IgG determined simultaneously on individual platelets. Platelet samples from 10 normals and 31 thrombocytopenic patients were studied. PA-IgG was estimated using immunofluorescent FITC anti-IgG antibody. Binding of FITC anti-IgG to the platelets was quantitated in the flow cytometer as relative mean fluorescence (RMF) which was calibrated against values (in fg/plt of FITC anti-IgG) obtained by spectrofluorometry after solubilization of the platelets. A high correlation (r = 0.89) was found between flow cytometric RMF value and spectrofluorometric FITC anti-IgG values. The flow cytometric studies showed that platelet samples with abnormally elevated levels of FITC anti-IgG (greater than 1.7 fg/plt) not only have a higher percentage of platelets with elevated FITC anti-IgG, but that these platelets also have increased levels of FITC anti-IgG as compared to platelets from normal samples. Platelet size was measured by the amount of forward light scatter in the flow cytometer. A low but significant correlation (r = 0.33 +/- 0.12) was found between size (FALS) and fluorescent signals in samples with elevated FITC anti-IgG. The contribution of 10% of the smallest platelets by FALS and 10% of the largest platelets by FALS to the total levels of flow cytometer platelet fluorescence in these samples was only 4.4% and 19.4% respectively which was not higher than obtained with samples with normal levels of FITC anti-IgG. In conclusion, this study showed that increased levels of PA-IgG found among thrombocytopenic patients were not confined to any particular size class of platelets.

Blood Platelets↗
PubMed1999

Evaluation of the hemostatic function of stored platelet concentrates using the platelet function analyzer (PFA-100 ).

BACKGROUND AND OBJECTIVE: Progressive functional impairment is known to occur in platelet concentrates through the storage period. Standardized methods providing direct measurement of residual platelet function in stored platelets are lacking. The purpose of this study was to determine whether a new platelet function analyzer (PFA-100 ) could provide standardized methods for assessing the hemostatic capacity of stored platelets. DESIGN AND METHODS: The PFA-100 was used to evaluate platelet function in stored platelets. The instrument can process citrated whole blood but it is unable to process platelet suspensions. Accordingly, the function of platelet concentrates should be measured following reconstitution of pseudo-whole blood. The analysis of the results included the closure time (sec) and a predictive index, an arithmetical index computed on the basis of the instrument's output data: the flow rate, the flow volume, the closure time. RESULTS: A final hematocrit of 58+/-2 and a final platelet concentration of 230+/-20x10(9)/L were used as standardized operative conditions to measure the function of stored platelet concentrates. The closure time (PFA-CT) and the predictive index (PFA-PI) both resulted to be capable of discriminating platelet concentrates with maintained or impaired function. PFA-PI was more informative than PFA-CT in terms of description of the residual platelet function. Of the two agonists used, epinephrine (EPI) resulted to be particularly sensitive for the detection of initial platelet hyporeactivity, whereas adenosine 5'-diphosphate (ADP) was particularly useful for measuring the residual platelet reactivity. INTERPRETATION AND CONCLUSIONS: PFA-CT and PFA-PI can be standardized; they provide new information about the hemostatic function of stored platelet concentrates and can be used to assess the quality of platelet concentrates.

Blood Platelets↗
PubMed1984

The interpretation of platelet kinetic studies for the identification of sites of abnormal platelet destruction.

The kinetics of platelets labelled with 111In have been studied in a series of 175 subjects including 18 normal volunteers, and 12 patients with idiopathic thrombocytopenic purpura (ITP), but excluding patients in whom there was scintigraphic evidence of intravascular platelet consumption. From analysis of the kinetics, the following parameters were calculated: splenic blood flow (SBF), intrasplenic platelet transit time (t-), splenic platelet pool capacity (expressed as a percentage of the total circulating platelet population), the fraction of the dose of labelled platelets ultimately destroyed in the spleen and the mean platelet life span (MPLS). SBF increased with increasing spleen size up to values of 25% total blood volume (TBV) per min. Some patients with immune complex related diseases were identified with elevated SBF (up to 24% TBV min-1) but without significant splenomegaly. Patients with cardiac decompensation had reduced SBF relative to spleen size. t- showed no relationship with spleen size. It tended to fall in patients who had high SBF relative to spleen size and to rise in those with low SBF relative to spleen size; i.e. it was inversely related to splenic perfusion (flow per unit tissue volume). The splenic platelet pool capacity is dependent on platelet input (SBF) and splenic platelet clearance (reciprocal of t-), and showed a close relationship with spleen size. When all subjects except those with ITP were considered, splenic platelet destruction showed a good correlation (r = 0.70, n = 42, P less than 0.001) with the splenic platelet pooling capacity. The ratio of the fraction of platelets destroyed in the spleen to the fraction pooling there, the D/P ratio, was approximately unity and did not appear to vary with MPLS, spleen size or the patient's condition, except in ITP where it varied between about 0.5 and 2. This variation in ITP was thought to be the result of an immune mediated re-direction of reticulo-endothelial platelet destruction. It is suggested that the D/P ratio, rather than the absolute quantity of 111In labelled platelets destroyed in the spleen, may be a more useful predictor of response to splenectomy since it takes into account the observed, appropriate, tendency for the spleen to destroy platelets in proportion to its platelet pooling capacity.

Blood Flow Velocity↗
PubMed1993

[Quality control of platelet concentrates. Functional assessment of stored platelets in vitro].

Platelet concentrates transfused for correction of thrombocytopenia or reduced platelet function do not consistently improve primary haemostasis in the recipient. Insufficient therapeutic effects may be caused by impaired donor platelet function and by unfavourable donation and storage conditions, as well as by immunological interactions with the recipient blood. The present study was designed to investigate whether the effect of platelet transfusion on recipient platelet function can be predicted by in vitro methods. METHODS. Blood samples were taken from 12 thrombocytopenic patients before (20 ml, P0) and after (10 ml, P(vivo)) transfusion of one unit of platelets previously stored for 24-120 h in acid citrate dextrose. An additional sample was taken from the platelet concentrate (TK) immediately before transfusion. P0 was divided into two specimens and TK platelets were added to one of them (P(vitro) in order to obtain a platelet count similar to that in P(vivo). Bleeding time (BT) and bleeding volume (BV) of the samples P0, P(vivo) and P(vitro) were measured using the method of Kratzer and Born (Fig. 2); mean values were calculated for each sample from six measurements. Aggregability of TK platelets was determined in addition by aggregometry. In contrast to previous studies, physiological Ca2+ concentrations were restored and secondary haemostasis was inhibited by low-molecular-weight heparin (Fragmin P, Pfrimmer Kabi GmbH und Co. KG, Erlangen) in the platelet-rich plasma used for aggregometry. RESULTS. Platelet counts increased in all patients after transfusion (P(vivo) vs P0, Table 1) and were nearly identical in P(vitro) and P(vivo) (r = 0.94, P < 0.001; Fig. 3). Parameters of primary haemostasis were significantly improved by addition of platelets to P0 in vitro (BT P < 0.05, BV P < 0.01) as well as by platelet transfusion (BT P < 0.05, BV P < 0.01). Direct comparison of P(vitro) and P(vivo) yielded a very close correlation of BT (r = 0.88, P < 0.001) and BV (r = 0.89, P < 0.01) in both samples. Although aggregometry revealed decreasing platelet function with increased storage time, aggregability was considerably higher compared to previous studies of platelet concentrates stored for 2-5 days. CONCLUSION. A new technique has been developed which allows reliable prediction of the effect of platelet concentrates on primary haemostasis of the recipient by in vitro measurement of bleeding time and bleeding volume prior to transfusion using the method of Kratzer and Born.

Blood Platelets↗
PubMed2005

Effects of SolCD39, a novel inhibitor of Platelet Aggregation, on Platelet Deposition and Aggregation after PTCA in a Porcine Model.

INTRODUCTION: This study evaluated CD39 in a porcine model of balloon angioplasty and in plasma of patients undergoing percutaneous intervention. CD39 (E-NTPDase1), is the endothelial ecto-ADPase inhibiting platelet function via hydrolysis of released platelet ADP. METHODS AND RESULTS: A recombinant soluble form of CD39 (solCD39) given intravenously to pigs had an elimination half life of 5--7 days, increased the bleeding time to an extent similar to aspirin, and inhibits platelet aggregation by>90%. Platelet counts and clot retraction remained normal following solCD39 administration. In a pig model of acute coronary balloon injury, solCD39 resulted in non-statistically significant decreases in platelet (7.7+/-1.4 versus 11.7+/- 3.4) and fibrin (3.5+/- 0.4 versus 4.2+/- 0.7) deposition ratios. Adding ex vivo to human platelet rich plasma (PRP) solCD39 produced nearly 100% inhibition of ADP-induced platelet aggregation. A dose-response effect of solCD39 on platelet aggregation induced by collagen or a thrombin receptor activating peptide (TRAP(SFLLRN)) was noted in PRP obtained from volunteers and patients receiving aspirin, clopidogrel or ticlopidine. SolCD39 also provided additional and complete inhibition of TRAP-induced platelet aggregation in PRP from patients who had received abciximab, aspirin and clopidogrel. CONCLUSIONS: SolCD39, a novel inhibitor of platelet activation and recruitment with a relatively long half-life appears to be well tolerated and is a potent inhibitor of ADP-, collagen-, or TRAP-induced platelet activation. Its potential use in percutaneous coronary intervention requires further study. ABBREVIATED ABSTRACT: E-NTPDase1/CD39 is the endothelial ecto-ADPase responsible for inhibition of platelet function. A recombinant soluble form (solCD39) had an elimination half life of 5-7 days in pigs, elevated bleeding times similar to aspirin, did not affect clot retraction, and inhibited platelet aggregation by > 90%. When combined with standard heparin therapy in a pig model of acute coronary balloon injury, solCD39 resulted in a trend toward a decrease in platelet and fibrin deposition. SolCD39 added ex vivo to human platelet rich plasma yielded nearly 100% inhibition of ADP-induced platelet aggregation and provided further inhibition when combined with standard therapy.

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