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PubMed1992

Endothelium-derived relaxing factor inhibits thrombin-induced platelet aggregation by inhibiting platelet phospholipase C.

Endothelium-derived relaxing factor (EDRF) inhibits platelet function, but the mechanism underlying this inhibitory effect is not known. To examine this, cultured acetylsalicylic acid (ASA)-treated endothelial cells (EC) from bovine aorta (BAEC) or from human umbilical vein (HUVEC) were incubated with washed, ASA-treated human platelets. Incubation of platelets with either BAEC or HUVEC resulted in inhibition of thrombin-induced platelet aggregation that was dependent on the number of EC added. This effect was potentiated by superoxide dismutase and reversed by treating EC with NG-nitro-L-arginine or by treating platelets with methylene blue, indicating that the inhibition of platelet aggregation was due to the release of EDRF by EC. EC significantly blocked the thrombin stimulated breakdown of phosphatidylinositol-4,5-bisphosphate (PIP2) and the production of phosphatidic acid in [32P]orthophosphate-labeled platelets and of inositol trisphosphate in [3H]myoinositol-labeled platelets. In addition, the thrombin-mediated activation of protein kinase C (PKC) and phosphorylation of myosin light chain were inhibited in the presence of EC. Finally, thrombin stimulated an increase in cytosolic ionized calcium concentration ([Ca2+]i) in fura2-loaded platelets that was abolished by concentrations of EC which also blocked thrombin-induced aggregation. These data indicate that EDRF blocks thrombin-induced platelet aggregation by inhibiting the activation of PIP2-specific phospholipase C and thereby suppressing the consequent activation of PKC and the mobilization of [Ca2+]i.

Animals↗
PubMed2005

[Study on platelet activated state and platelet activated function in adults with acute leukemia].

To investigate the changes of platelet activated state and platelet activated function by trace whole blood flow cytometry (FCM), and to explore the mechanism of hemorrhage and infiltration in adults with acute leukemia, the expression percentage and changes of these expressions of CD62p and PAC-1 on platelet surface were determined by FCM of trace whole blood after platelet activated by ADP in patients with new diagnosed AL (group I), complete remission (CR, group II) and continuously complete remission (CCR, group III). Healthy adults were used as control group. The result showed that the expression of CD62p in group I and II was higher than that in control group, before and after platelet activated by ADP (P < 0.01). The expression of PAC-1 in group I was higher than that in control group (P < 0.01), the expression of PAC-1 in group II was lower than that in control group (P > 0.01), There was no significant difference in expression of CD62p and PAC-1 between group III and control group (P > 0.01), and no significant difference was found between AL group with megakaryocyte malignant pathological changes and AL group without megakaryocyte malignant pathological changes before platelet activated by ADP (P > 0.01). After platelet activated by ADP, the expression of PAC-1 in the former was lower than that in the latter (P < 0.01). It is concluded that (1) high level activated platelet in peripheral blood of AL patients show that interaction between activated platelet and leukemia cells can be one of reason resulting in widespread hemorrhage and infiltration AL patiens; (2) the decrease of number and activted function of platelet at the first stage of AL patients may be caused by malignant hyperplasia of leukemia cells and damage of megakaryopoiesis in bone marrow.

Acute Disease↗
PubMed2006

Autologous platelet gel: an in vitro analysis of platelet-rich plasma using multiple cycles.

Autologous platelet gel (APG) has become an expanding field for perfusionists. By mixing platelet-rich plasma (PRP) with thrombin and calcium, platelet gel is prepared and used in many surgical settings. There are many devices used to produce PRP. This study evaluates the Medtronic Magellan Autologous Platelet Separator. The purpose of this study was to show that processing two cycles of the same syringe could reduce the amount of blood required to produce a specific volume of PRP. Three 60-mL syringes of whole blood with anticoagulant were removed from 15 elective coronary artery bypass patients. Each syringe produced 9 mL of PRP and 1 mL was sent to the laboratory for analysis. The remaining whole blood in each syringe was processed a second time with a yield of 5 mL of PRP with 1 mL sent to the laboratory. With this data, the Magellan was assessed in three phases. The first phase focused on the consistency of the Magellan. Laboratory values of hematocrit, platelet count, white blood cell count, and fibrinogen were compared between each syringe processed by the device. The second phase dealt with the percentage of platelets in the PRP that the Magellan was able to capture. Finally, results of both cycles were combined and compared against baseline values. Most of the hematological factors evaluated between each syringe were consistent in both cycles. The Magellan was able to capture nearly 70% of all platelets in the PRP of the first cycle and 18.5% in the second cycle. By mathematically combining both cycles, platelet counts averaged 2.8 times baseline with a 3.3 times baseline increase when the volume of the two cycles was weighted. This weighted average was done to reflect a higher concentration of Cycle 1 platelets than Cycle 2 in each sample. This study proved that processing each syringe of whole blood twice could reduce blood requirements while maintaining an effective platelet yield and volume. It also showed that the Magellan does conform to benchmark testing done at Medtronic.

Blood Platelets↗
PubMed1990

Effect of platelet activation inhibitors on the loss of glycoprotein Ib during storage of platelet concentrates.

Platelet membrane glycoprotein Ib (GPIb) in stored platelet concentrates was analyzed by flow cytometry with three separate monoclonal antibodies (AN-51, 6D1, and SZ-2), by tritiated glycoprotein radiolabeling, and by ristocetin-induced agglutination. Flow cytometry showed that a population of surface GPIb-negative platelets was evident at 5 days and increased three- to fivefold by the tenth day. Tritium radiolabeling of surface GPIb showed a decrease over 10 days of 37% +/- 17%. The degree of loss of surface GPIb correlated well with other changes during storage: decreased ristocetin-induced agglutination, decreased responsiveness in the hypotonic shock test, lower plasma pH, and increased extracellular lactic dehydrogenase. Immunoblotting of total platelet GPIb with the SZ-2 antibody showed a decrease of 58% +/- 16% during the 10-day storage period. The effect of protease inhibitors or platelet activation inhibitors on the loss of GPIb during storage was studied in platelet concentrates paired with untreated controls. Only the platelet activation inhibitors prostaglandin E1 and theophylline retarded the loss of surface GPIb levels (93% +/- 5% GPIb remaining vs 65% +/- 16%). Total GPIb levels also decreased less in the presence of the activation inhibitors (45% +/- 22% lost vs 70% +/- 14% lost). These findings suggest that platelet activation, rather than plasma enzymatic activity, is responsible for the loss of platelet GPIb during storage of platelet concentrates.

Alprostadil↗
PubMed1989

Inhibition of platelet activating factor-induced platelet aggregation by molsidomine, SIN-1, and nitrates in vitro and ex vivo.

We compared in vitro the effects of molsidomine, its active metabolite SIN-1, sodium nitroprusside, and the organic nitrates nitroglycerin, isosorbide-5-mononitrate, and isosorbide-2,5-dinitrate on platelet aggregation induced by platelet activating factor and on the activity of soluble guanylate cyclase. In addition, the effects of molsidomine and of isosorbide-5-mononitrate on ex vivo platelet function were studied. In vitro, SIN-1 and sodium nitroprusside were about 100-fold more potent activators of platelet guanylate cyclase and inhibitors of platelet activating factor-induced aggregation than the other agents. In contrast, in ex vivo experiments, not only molsidomine but also isosorbide-5-mononitrate inhibited platelet activating factor-induced aggregation. These data indicate that molsidomine, SIN-1, and organic nitrates can in vivo, like endothelium-derived relaxing factor, inhibit platelet aggregation and exert antithrombotic properties, although nitrates apparently cannot be converted in platelets to active metabolites. Since the antiaggregatory properties are observed when platelet activating factor is used as an aggregant, and since platelet activating factor-induced aggregation is only weakly influenced by inhibitors of cyclo-oxygenase, this effect might be useful clinically.

Cyclic GMP↗
PubMed1989

Inhibition of platelet-von Willebrand factor binding to platelets by adhesion site peptides.

Synthetic peptides containing the adhesion site recognition sequences present on the A alpha and gamma chains of fibrinogen were studied for their effect on the binding of endogenous platelet-von Willebrand factor (vWF) and exogenous plasma-vWf to thrombin-stimulated platelets. In agreement with previously reported data, the tetrapeptide consisting of the RGDS sequence was a more potent inhibitor of plasma-vWf binding to platelets than was the pentadecapeptide of the carboxy terminus of the fibrinogen gamma-chain (IC50 10.6 mumol/L for the RGDS tetrapeptide v 44.9 mumol/L for the gamma-chain pentadecapeptide). No apparent synergy in the inhibition of plasma-vWf binding was noted when the RGDS and gamma-chain peptides were used together (IC50 15.2 mumol/L). In contrast, the gamma-chain peptide was significantly more inhibitory than was the RGDS tetrapeptide on the binding of platelet-vWf to platelets (IC50 1.4 mumol/L for the gamma-chain pentadecapeptide v 4.5 mumol/L for the RGDS tetrapeptide, P less than .05), and there was significant synergy in the inhibition of platelet-vWf binding noted when the gamma-chain and RGDS peptides were used together (IC50 0.04 mumol/L). These results indicate that the binding of platelet-vWf to its receptor on the platelet glycoprotein IIb/IIIa complex involves both the RGDS and gamma-chain recognition sites. In contrast to the results with plasma-vWf binding, the gamma-chain recognition site appears to be more important than the RGDS recognition site in platelet-vWf binding to platelets.

Binding, Competitive↗
PubMed1989

Rapid purification and partial characterization of human platelet glycoprotein IIIb. Interaction with thrombospondin and its role in platelet aggregation.

Glycoprotein IIIb (also known as glycoprotein IV) is a major glycoprotein present on the surface of human platelets. Recent studies suggest that glycoprotein IIIb may be a receptor site for thrombospondin. Thrombospondin, a multifunctional adhesive glycoprotein released from stimulated platelets, plays an important role in the stabilization of platelet aggregates. In this study, a new method for the purification of glycoprotein IIIb is described. Glycoprotein IIIb was isolated from Triton X-114 platelet membrane extracts, under nondenaturing conditions, by tandem anion-exchange and size exclusion fast protein liquid chromatography. The purified glycoprotein had the same apparent molecular mass (88 kDa) under nonreducing or reducing conditions. The tryptic peptide map of the purified protein was identical to that of bona fide glycoprotein IIIb as isolated from two-dimensional polyacrylamide gels of platelet membrane proteins. In addition, the purified glycoprotein was recognized by an anti-GPIIIb monoclonal antibody (OKM5). The purified glycoprotein specifically bound to thrombospondin in the presence of calcium. Monospecific anti-GPIIIb antibodies interfered with the expression of endogenous thrombospondin on thrombin-activated platelets and partially inhibited collagen- and thrombin-induced platelet aggregation without a significant effect on platelet secretion. Glycoprotein IIIb, by interacting with thrombospondin on the activated platelet surface, may play an important role in the platelet aggregation process.

Blood Platelets↗
PubMed1985

Preliminary functional characterization of a 24,000 dalton platelet surface protein involved in platelet activation.

We have recently investigated three monoclonal antibodies, FMC 8, FMC 48 and FMC 56, directed against a 24,000 dalton (24K) cell surface protein on human platelets and pre-B progenitor cells. These monoclonal antibodies (at 0.1-10 micrograms/ml) were found to be potent inducers of platelet aggregation with normal and with aspirin-treated platelets. FMC 8 Fab' and FMC 56 (Fab')2 fragments blocked aggregation induced by intact antibody. The Fab' and (Fab')2 fragments, however, were ineffective by themselves (at 100 micrograms/ml) as platelet agonists but augmented (at 10-25 micrograms/ml) the aggregation response of normal and aspirin-treated platelets to threshold concentrations of ADP. It has been suggested that the 24K cell surface protein may be identical to a 24K cAMP-dependent phosphoprotein involved in regulating transmembrane calcium flux. To examine this, 32P-phosphate-loaded platelets were treated with prostaglandin E1 to raise intracellular cAMP levels and then analysed on two-dimensional O'Farrell gels. No labelled phosphoprotein corresponded in mobility to the 24K cell surface protein. Further, FMC 56 failed to immunoprecipitate a 24K phosphoprotein. The combined results suggest that the three monoclonal antibodies activate platelets via a prostaglandin-independent and as yet undefined mechanism which requires the intact bivalent antibody for full platelet response. Fab' fragments (monovalent) and (Fab')2 fragments (altered Fab-Fab configuration) are ineffective as platelet agonists but can augment the response of other stimuli. Thus, further studies with this system should allow delineation of those intracellular events necessary for partial and full platelet responses.

Antibodies, Monoclonal↗
PubMed1985

The tetrapeptide analogue of the cell attachment site of fibronectin inhibits platelet aggregation and fibrinogen binding to activated platelets.

Fibrinogen binding to receptors on activated platelets is a prerequisite for platelet aggregation. However, the regions of fibrinogen interacting with these receptors have not been completely characterized. Fibronectin also binds to platelet fibrinogen receptors. Moreover, the amino acid sequence Arg-Gly-Asp-Ser, corresponding to the cell attachment site of fibronectin, is located near the carboxyl-terminal region of the alpha-chain of fibrinogen. We have examined the ability of this tetrapeptide to inhibit platelet aggregation and fibrinogen binding to activated platelets. Arg-Gly-Asp-Ser, but not the peptide Arg-Gly-Tyr-Ser-Leu-Gly, inhibited platelet aggregation stimulated by ADP, collagen, and gamma-thrombin without inhibiting platelet shape change or secretion. At a concentration of 60-80 microM, Arg-Gly-Asp-Ser inhibited the aggregation of ADP-stimulated gel-filtered platelets approximately equal to 50%. Arg-Gly-Asp-Ser, but not Arg-Gly-Tyr-Ser-Leu-Gly, also inhibited fibrinogen binding to ADP-stimulated platelets. This inhibition was competitive with a Ki of approximately equal to 25 microM but was incomplete even at higher tetrapeptide concentrations, indicating that Arg-Gly-Asp-Ser is a partial competitive inhibitor of fibrinogen binding. These data suggest that a region near the carboxyl-terminus of the alpha-chain of fibrinogen interacts with the fibrinogen receptor on activated platelets. The data also support the concept that the sequence Arg-Gly-Asp-Ser has been conserved for use in a variety of cellular adhesive processes.

Adenosine Diphosphate↗
PubMed1987

Inhibitory effect of trichothecene mycotoxins on bovine platelets stimulated by platelet activating factor.

Several species of fungi, which infect cereals and grains, can produce a class of compounds, known as trichothecene mycotoxins, which is characterized by a substituted epoxy-trichothecene ring structure. Cattle are susceptible to intoxication from feeds contaminated with T-2 toxin, one of the more potent trichothecene mycotoxins, while swine refuse to ingest feed contaminated with T-2 toxin. The bovine platelet has been used as a model cell system to evaluate the effects of T-2 toxin and its natural metabolites, HT-2 toxin and T-2 tetraol, on cell function in vitro. Due to the lipophilic nature of these mycotoxins, a biologically active phospholipid was used to stimulate the platelets in the presence and absence of the toxins. The mycotoxin T-2 toxin and its major metabolite HT-2 toxin inhibited platelet activating factor-stimulated bovine platelets, suspended in homologous plasma, in a concentration but not time dependent manner. Significant inhibition of platelet function (p less than 0.01) occurred with 135 ng T-2 toxin per 10(6) platelets and with 77 ng HT-2 toxin per 10(6) platelets. These mycotoxins exerted an additive inhibitory effect on the platelet aggregation response. In contrast, the minor metabolite T-2 tetraol had no inhibitory effect on platelet function and had no influence on the responses of T-2 toxin or HT-2 toxin when the mycotoxins were present together in the platelet suspensions.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Platelet size and mass as an indicator for platelet transfusion after cardiopulmonary bypass.

Platelet count, mean platelet volume (MPV), and plateletcrit (PCT) were studied in 51 patients after cardiopulmonary bypass (CPB). MPV was significantly lower in 10 patients who developed postoperative bleeding (bleeders) compared to 41 with no significant bleeding (nonbleeders) (7.7 +/- 0.86 vs 8.5 +/- 1.2 fl, p less than .05). Postoperative platelet count was significantly lower in the group of bleeders (93.3 +/- 22.4 vs 127.5 +/- 43 X 10(9)/liters, p less than .02). A cutoff point of MPV or platelet count that would include bleeders and exclude nonbleeders could not be found due to the large overlap between the two groups. However, such a cutoff point does exist for PCT (PCT = total platelet mass). PCT was significantly lower among the bleeders (0.072 +/- 0.02% vs 0.108 +/- 0.036%, p less than .05) and a cutoff point of PCT less than 0.1% included all the bleeders and excluded 65% of nonbleeders. The low PCT and bleeding tendency can be corrected by platelets transfusion. In 15 patients (eight bleeders and seven nonbleeders) with low postoperative PCT (0.078 +/- 0.014), transfusion of 10 platelet units increased platelet count from 101 +/- 32 to 169 +/- 22 X 10(9)/liter, increased PCT to 0.128 +/- 0.2%, and stopped bleeding in all bleeders. A finding of PCT less than 0.1% after CPB is a clear indication for platelet transfusion in patients who develop post-CPB bleeding. This supports the observation that large platelets are more active than smaller ones, and that PCT, rather than PLT counts, predicts the risk of bleeding in patients with thrombocytopenia.

Adolescent↗
PubMed1985

Ultrastructural evidence for extravascular platelet recruitment in the lung upon intravenous injection of platelet-activating factor (PAF-acether) to guinea-pigs.

Bronchoconstriction and degenerative lesions of the bronchial epithelium were observed microscopically 1 min after the i.v. injection of 100 ng/kg of platelet-activating factor (PAF-acether) to the anaesthesized guinea-pig. Constricted arterioles containing marginated polymorphonuclear neutrophils and platelet aggregates were seen, as well as alveolar capillaries obstructed by thrombi formed by partially or totally degranulated platelets. Three minutes after the injection of PAF-acether, platelet diapedesis to the alveolar septa and lumen was clearly observed. Bronchoconstriction was still present at 3 min, but subsided after 60 min, whereas oedema of the submucosa persisted accompanied by an infiltration of eosinophils and neutrophils. The infusion of prostacyclin prevented the formation of platelet aggregates and platelet diapedesis due to PAF-acether, but the morphological evidence of bronchial constriction was not modified. Aspirin failed completely to modify the effects of PAF-acether. Our results show that PAF-acether causes early formation and deposition of platelet aggregates, accompanied by the margination of polymorphonuclear neutrophil leucocytes in pulmonary vessels of the guinea-pig. Since bronchoconstriction persisted when platelet aggregation was inhibited with prostacyclin, aggregation by itself would not account for this effect. Early platelet diapedesis in the vicinity of bronchial smooth muscle corroborates previous evidence that platelets contain and release bronchoconstrictor substances, which operate by cyclo-oxygenase-independent mechanisms and are possibly involved with the physiopathology of lung inflammation during immediate hypersensitivity.

Adenosine Diphosphate↗
PubMed1974

Concentration effects of platelets, fibrinogen and thrombin on platelet aggregation and fibrin clotting.

The effects of varying concentrations of platelets, fibrinogen and thrombin on platelet aggregation and on fibrin clotting were investigated. The results indicated that a threshold thrombin to platelet concentration ratio may be required to cause platelet activation. Above the threshold ratio, platelets exhibited properties which enhanced thrombin action in causing aggregation and fibrin clotting. At T/P ratios below the threshold level, the presence of platelets reduced thrombin activity, in other words, platelets exerted an antithrombin action. Fibrinogen at low concentrations (0.02-1.5 mg/ml) enhanced platelet aggregation induced by thrombin; whereas, at high concentrations of fibrinogen (2.0-4.0 mg/ml), aggregation was markedly inhibited. Continuous mixing of samples of paltelets and fibrinogen at physiological concentrations with thrombin at low concentrations (less than 2.0 U/ml) resulted in platelet aggregation. On the other hand, fibrin clots formed in samples without mixing or with high thrombin concentrations (greater than or equal to 5.0 U/ml). These results suggested that the quantitative relationships between platelets, fibrinogen and thrombin, and the presence or absence of cell contact may be important factors in determining the overall hemostasis.

Antithrombins↗
PubMed1984

In vivo interaction of von Willebrand factor with platelets following cryoprecipitate transfusion in platelet-type von Willebrand's disease.

Previous studies performed in vitro have indicated that platelets from patients with platelet-type von Willebrand's disease (vWD) have receptors for von Willebrand factor (vWF) already exposed on their surfaces and that the addition of purified vWF or cryoprecipitate to patient platelet-rich plasma under stirring conditions is capable of inducing platelet aggregation and secretion. The present work reports the results of the transfusion of cryoprecipitate in a patient with platelet-type vWD. It is shown that, while factor VIII-related antigen and ristocetin cofactor activities maintain elevated levels for up to 12 hr following transfusion, the highest molecular weight vWF multimers decline rapidly. The platelet count also declines, followed in turn by a rise in the plasma level of platelet factor 4. Shortening of the bleeding time occurs only very transiently. The results of this study provide direct evidence that, in patients with platelet-type vWD, an abnormal interaction of their platelets with plasma vWF occurs in vivo, resulting in the absence of high molecular weight vWF multimers, low platelet counts, and impaired hemostasis that are characteristic of this disease.

Adult↗
PubMed1981

Gray platelet syndrome: alpha-granule deficiency. Its influence on platelet function.

The ultrastructure, cytochemistry, biochemistry, and functions of platelets from two patients with the familial gray platelet syndrome are described. Ultrastructural studies showed a lack of alpha-granules in the megakaryocytes in the vicinity of a marrow myelofibrosis and/or in platelets. A normal number of mitochondria and a slight increase of dense bodies was confirmed by labeling the whole patient with mepacrine. Platelet peroxidase (in the dense tubular system) and catalase-positive granules (revealed by the cytochemistry) were present. Platelets from both patients had severe deficiency of beta TG either after tritonization (less than 4% of normal) or thrombin treatment (less than 15% of normal), and the plasma beta TG levels were slightly increased. Functional studies of these platelets showed an uptake of [14C]5HT inhibited by reserpine similar to that in the control platelets. Thromboxane formation was within normal limits in the presence of arachidonic acid, ADP, collagen, or ionophore A23187, indicating that (1) the cyclooxygenase/thromboxane synthetase systems were not altered and (2) the phospholipase activities were not impaired. Although the platelet adhesion to prepolymerized fibrillar type III collagen and the ADP-, arachidonic acid-, and ionophore A23187-mediated aggregations were apparently normal, in every case the release of [14C]5HT was either at the low side of the normal range or decreased. This abnormality was increased when collagen or thrombin was added to either PRP or washed platelets from both patients, where at the same time, the aggregation and the release were markedly reduced. The results suggest that alpha-granules or their content has an influence on not only the platelet aggregation but also the dense bodies involved in the [14C]5HT release reaction.

Adolescent↗
PubMed1980

A comparison of the effects of dietary short and long chain saturated fatty acids on platelet functions, platelet phospholipids, and blood coagulation in rats.

Rats were fed for 20 weeks balanced purified diets solely enriched in fat, to determine whether, as in diets containing cholesterol and cholic acid, it was mostly the common dietary long chain saturated fatty acids (16:0 and 18:0) which could induce platelet function abnormalities. The dietary fats rich in 16:0 and 18:0 induced increased thrombin and adenosine diphosphate (ADP) platelet aggregation and an acceleration of the platelet-rich plasma clotting time. These effects on platelet functions were coupled with increased levels of 20:3 omega 6 and 20:3 omega 9 in total platelet phospholipids and a decrease of 22:4 and 24:4 omega 6 in the platelet phosphatidyl ethanolamine. Fatty acids that most closely correlated with thrombin aggregation and plasma clotting time (r = 0.99) were 20:3 omega 9 and 22:4 omega 6. There was no relationship between the effects of dietary fats on blood lipids and on blood platelets. Consequently, in different experimental models in rats, the dietary long chain saturated fatty acids appear to be responsible for certain platelet function abnormalities (clotting activity, response to thrombin aggregation). These platelet abnormalities might be due to changes in the platelet phospholipid fatty acid composition.

Adenosine Diphosphate↗
PubMed1995

Regulation of platelet aggregation by post-fibrinogen binding events. Insights provided by dithiothreitol-treated platelets.

A variety of data suggest that fibrinogen binding is necessary but not sufficient for platelet aggregation: post fibrinogen binding events may play an important role. The present study compared fibrinogen binding and platelet aggregation in response to dithiothreitol (DTT) and ADP. DTT induced saturable and specific fibrinogen binding (Kd 0.07 + 0.02 microM, Bmax 15,000 + 3000 molecules/platelet) which supported complete platelet aggregation as determined by single platelet counting. The aggregates were small, however, and more readily dissociated by EDTA than their ADP-treated counterparts, despite quantitatively similar fibrinogen binding. Unlike fibrinogen bound to ADP-stimulated platelets, fibrinogen bound to DTT-treated platelets remained sensitive to dissociation by EDTA over a 3 h time course, retained its ability to support aggregation, even when aggregation was induced 60 min after the initial platelet exposure to fibrinogen, and remained accessible to polyclonal antibodies and plasmin. Confocal scanning laser microscopy showed only a surface clustering of fibrinogen bound to DTT-treated platelets over the 3 h time course compared to rapid fibrinogen clearing from the surface of ADP-stimulated platelets. These data suggest that post fibrinogen binding events involved in the stabilization of fibrinogen binding and/or the redistribution of bound fibrinogen may play important roles in regulating platelet aggregation.

Adenosine Diphosphate↗
PubMed1995

The value of flow cytometric analysis of platelet glycoprotein expression of CD34+ cells measured under conditions that prevent P-selectin-mediated binding of platelets.

In the present study, we show by adhesion assays and ultrastructural studies that platelets can bind to CD34+ cells from human blood and bone marrow and that this interaction interferes with the accurate detection of endogenously expressed platelet glycoproteins (GPs). The interaction between these cells was found to be reversible, dependent on divalent cations, and mediated by P-selectin. Enzymatic characterization showed the involvement of sialic acid residues, protein(s). The demonstration of mRNA for the P-selectin glycoprotein ligand 1 (PSGL-1) in the CD34+ cells by polymerase chain reaction (PCR) analysis suggests that this molecule is present in these cells. Under conditions that prevent platelet adhesion, a small but distinct subpopulation of CD34+ cells diffusely expressed the platelet GPIIb/IIIa complex. These cells were visualized by immunochemical studies. Furthermore, synthesis of mRNA for GPIIb and GPIIIa by CD34+ cells was shown using PCR analysis. The semiquantitative PCR results show relatively higher amounts of GPIIb mRNA than of PF4 mRNA in CD34+CD41+ cells in comparison with this ratio in platelets. This finding is a strong indication that the PCR results are not caused by contaminating adhering platelets. MoAbs against GPIa GPIb alpha, GPV, P-selectin, and the alpha-chain of the vitronectin receptor did not react with CD34+ cells. The number of CD34+ cells expressing GPIIb/IIIa present in peripheral blood stem cell (PBSC) transplants was determined and was correlated with platelet recovery after intensive chemotherapy in 27 patients. The number of CD34+CD41+ cells correlated significantly better with the time of platelet recovery after PBSC transplantation (r = .83, P = .04) than did the total number of CD34+ cells (r = .55). Statistical analysis produced a threshold value for rapid platelet recovery of 0.34 x 10(6) CD34+CD41+ cells/kg. This study suggests that if performed in the presence of EDTA the flow cytometric measurement of GPIIb/IIIa on CD34+ cells provides the most accurate indication of the platelet reconstitutive capacity of the PBSC transplant.

Antigens, CD34↗