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PubMed2003

Development of platelet contractile force as a research and clinical measure of platelet function.

This article reviews work performed at the Medical College of Virginia of Virginia Commonwealth University during the development of a whole-blood assay of platelet function. The new assay is capable of assessing platelet function during clotting and thus allows measurement of the contribution of platelets to thrombin generation. Because platelets are monitored in the presence of thrombin, the test gages platelets under conditions of maximal activation. Three parameters are simultaneously assessed on one 700 microL sample of citrated whole blood. Platelet contractile force (PCF), the force produced by platelets during clot retraction, is directly measured as a function of time. This parameter is sensitive to platelet number, platelet metabolic status, glycoprotein IIb/IIIa status, and the presence of antithrombin activities. Clot elastic modulus (CEM), also measured as a function of time, is sensitive to fibrinogen concentration, platelet concentration, the rate of thrombin generation, the flexibility of red cells, and the production of force by platelets. The third parameter, the thrombin generation time (TGT) is determined from the PCF kinetics curve. Because PCF is absolutely thrombin dependent (no thrombin-no force), the initial upswing in PCF occurs at the moment of thrombin production. TGT is sensitive to clotting factor deficiencies, clotting factor inhibitors, and the presence of antithrombins, all of which prolong the TGT and are known to be hemophilic states. Treatment of hemophilic states with hemostatic agents shortens the TGT toward normal. TGT has been demonstrated to be shorter and PCF to be increased in coronary artery disease, diabetes mellitus, and several other thrombophilic states. Treatment of thrombophilic states with a variety of heparin and nonheparin anticoagulants prolongs the TGT toward normal. The combination of PCF, CEM, and TGT measured on the same sample may allow rapid assessment of global hemostasis and the response to a variety of procoagulant and anticoagulant medications.

Animals↗
PubMed2001

Impaired platelet function among platelet donors.

BACKGROUND: Platelet transfusions are effective for the prevention and treatment of bleeding in patients with disorders of platelet number and/or function. In recent years plateletpheresis concentrates have outnumbered pooled platelet concentrates, albeit with significant differences between nations. Thus, the platelet quality of individual donors has become increasingly important. The aim of this study was to gain an estimate for the prevalence of impaired platelet function among platelet donors. STUDY DESIGN AND METHODS: We determined the inter-donor variability in platelet plug formation with a PFA-100 analyzer, the prevalence of impaired thromboxane formation, and effects of the density in alpha2 integrin polymorphism and density. RESULTS: (i) Collagen-epinephrine induced closure time (CEPI-CT) showed a great inter-subject variability in platelet donors and was higher than in healthy controls (p = 0.008). One-fifth of donors had abnormal CEPI-CT values and 11% exceeded >300 s (max measurable value). (ii) Decreased serum thromboxane B2 levels were found in 9% of all donors, compatible with surreptitious intake of cyclooxygenase inhibitors or with an aspirin-like defect. CEPI-CT correlated inversely with TxB2-levels in donors and controls. (iii) The density of the alpha2-integrin correlated negatively with CEPI-CT and CADP-CT values in controls, but was not responsible for the observed impaired platelet function in donors. (iv) Finally, the ABO blood group system modulates closure times. CONCLUSION: In sum, a large number of platelet donors present with prolonged closure times. Decreased thromboxane formation and frequent platelet donation partly account for this observation.

Adenosine Diphosphate↗
PubMed1998

Effects of acute myelogenous leukemia blasts on platelet release of soluble P-selectin and platelet-derived growth factor.

Complex interactions occur between platelets and normal as well as leukemic myeloid cells. In vitro co-culture of platelets and acute myelogenous leukemia (AML) blasts with allogeneic platelets enhances blast proliferation and constitutive cytokine secretion. In the present study the effects of AML blasts on the platelet release of soluble mediators are characterized. Normal platelets released soluble (s) P-selectin and platelet-derived growth factor (PDGF), both when cultured alone and in the presence of AML blasts, and for certain patients the presence of AML blasts increased the platelet release of these mediators. Addition of exogenous interleukin (IL) 10 to platelet-AML blast cultures further increased platelet release of PDGF and sP-selectin. For certain patients decreased AML blast cytokine secretion was observed when PDGF-specific antibodies were added to cultures with blasts plus platelets, these results indicate that platelet release of PDGF is a molecular mechanism for the enhancement of AML blast cytokine secretion. We conclude that complex functional alterations are induced both in AML blasts and normal allogeneic platelets during in vitro co-culture of leukemia cells and platelets.

Journal Article↗
PubMed1990

Cooperation between platelets and neutrophils for paf-acether (platelet-activating factor) formation.

Association of platelets and neutrophils is frequently observed within thrombi or inflammatory sites. Interactions between these two cell populations have been reported for the production of several mediators of inflammation such as hydrogen peroxides or leukotrienes. Another potential mediator of thrombosis and inflammation is paf-acether, which is synthesized by activated platelets and neutrophils. Since platelets form and release large amounts of the paf-acether precursor lyso paf-acether, platelet and neutrophil cooperation for paf-acether biosynthesis was investigated. Purified human neutrophils (4 x 10(6)/ml) stimulated by opsonized zymosan (ZC, 1 mg/ml) formed 4.5 +/- 2.5 ng/ml paf-acether. Human washed platelets (3 x 10(8)/ml) stimulated with thrombin (1 IU/ml) formed 0.60 +/- 0.43 ng/ml paf-acether. Platelets and neutrophils, incubated together and both stimulated by their specific agonist, formed more than twice as much paf-acether as did platelets and neutrophils separately (10.90 +/- 4.25 ng/ml, n = 6, P less than .001). The formation of lyso paf-acether and the release of lysozyme and LDH were unchanged under the cooperation conditions. The formation of paf-acether almost doubled (10.24 +/- 3.81 ng/ml paf-acether vs. 5.30 +/- 2.23, P less than .05, n = 4) when ZC-stimulated neutrophils were incubated with supernatants from thrombin-stimulated platelets as well as with synthetic lyso paf-acether. Extracted and purified lyso paf-acether from thrombin-stimulated platelets led to an increase of biosynthesis of paf-acether by neutrophils (13.86 +/- 2.26 ng/ml paf-acether vs. 5.76 +/- 0.38, P less than .05, n = 3). These results indicate that a cooperation between platelets and neutrophils exists for paf-acether formation. The phenomenon depends on a platelet-derived soluble factor, possibly lyso paf-acether. This cell-to-cell interaction is of interest since paf-acether is formed by and acting on platelets and neutrophils and represents a molecular basis for potent amplification of inflammatory reactions.

Animals↗
PubMed1997

Cytokine mRNA expression in human platelets and a megakaryocytic cell line and cytokine modulation of platelet function.

Platelet formation and function are regulated, in vivo, to varying degrees by cytokines in the micro-environment. While white blood cells are the major source of cytokines within the cardiovascular system, the question addressed in this study was whether platelets and the platelet precursor, the megakaryocyte, may also serve as a source of cytokines. Cytokines produced by or carried within platelets could be released at sites of vascular injury and participate in wound healing. Platelets and a human megakaryocyte-like cell line, HU3, were found to express message for interleukin 7 (IL-7), stem cell factor (SCF), transforming growth factor beta (TGF-beta), cMpl, the IgE receptor subunits Fc epsilon RI alpha gamma and the transcription factor, NF-E2. Other cytokines expressed in HU3 cells but not in platelets included IL-1 beta, IL-6, IL-10, IL-13, TNF-alpha and the FC epsilon RI beta subunit. The HU3 cell line seemed to be further along the maturation/differentiation pathway to platelet formation than a second blood derived bipotential cell line, MB02. The MB02 cell line did not express IL-6, IL-10, SCF, TNF-omega nor cMpl. Furthermore, culturing the HU3 cells in TPO appeared to repress expression of Fc epsilon RI beta directing the cell closer to the platelet phenotype. In light of the presence of cytokine expression in platelets/megakaryocytes, agonist-induced platelet aggregation was measured in the presence of added cytokines as a means to evaluate potential cytokine modulation of platelet function. Collagen-induced aggregations were significantly enhanced by IL-6, SCF and TPO. Other cytokines tested significantly stimulated the thrombin receptor activating peptide, SFLLRNP-, U46619- and ADP-induced platelet aggregations with TPO being the most consistent activator. It is possible that cytokines released from platelets act in concert with cytokines released from other cellular sources to modulate haemostasis and thrombosis differentially depending upon the site of injury.

Base Sequence↗
PubMed1995

p32, a platelet autoantigen recognized by an SLE-derived autoantibody that inhibits platelet aggregation.

We have previously described an SLE-derived human hybridoma autoantibody, 9604, which binds to activated but not to resting platelets, inhibits platelet aggregation, and immunoprecipitates a surface-labelled poly-peptide of 32,000 molecular weight (MW) (p32). In the present study, using a murine monoclonal anti-p32 antibody (8E8), we show that p32 is responsible for the binding of 9604 to activated platelets. First, 8E8 bound to the surface of activated but not resting platelets, as detected by flow cytometry, showing saturation with 1900 antibody molecules bound/platelet and a Kd of 72 nM. Second, complete inhibition of the binding of 9604 to ADP-activated platelets by 8E8 and inhibition of the reactivity of 8E8 with p32 by 9604 demonstrated that 8E8 and 9604 recognize the same 32,000 MW platelet polypeptide. Isolation of platelet proteins, using an 8E8-affinity column, showed high MW bands corresponding to multimers of p32 under non-reducing conditions, and isolated p32 tended to self-associate even under reducing conditions. High performance liquid chromatography (HPLC) demonstrated that the native p32-containing protein has an approximate MW of 450,000. A comparison of the functional properties of murine monoclonal 8E8 and human monoclonal 9604 revealed that both antibodies are cytotoxic to platelets in 51Cr-release assays. In contrast, 8E8, unlike 9604, did not affect platelet aggregation, suggesting that different epitopes may be recognized by the two antibodies. These findings demonstrate that p32 is a subunit of an activation marker that is expressed on the surface of activated platelets and recognized by an SLE-derived anti-platelet autoantibody. Our data also indicate that murine monoclonal antibodies produced against platelet autoantigens may not mimic the functional activities of spontaneously occurring autoantibodies, probably due to the recognition of different epitopes.

Animals↗
PubMed1994

The effects of doxazosin on platelet aggregation, platelet adhesion and blood coagulation in cynomolgus monkeys.

The effect of doxazosin, a selective alpha-1 adrenergic inhibitor, on hemostasis was investigated in 9 cynomolgus monkeys. During 12 weeks of doxazosin treatment (1 mg/kg per day), serum lipids, lipoprotein cholesterols, blood coagulation, platelet aggregation and template bleeding times were measured and compared with predrug values. In addition, platelet adhesion to cultured human umbilical vein endothelial cells (HUVEC) in the presence or absence of doxazosin was evaluated. Platelet aggregation was also determined in monkeys following chronic oral exposure to aspirin (162 mg/day). Doxazosin administration was associated with significant reductions in serum total cholesterol (TC) (-16%) and low density lipoprotein cholesterol (LDL-C) (-23%), while high density lipoprotein cholesterol (HDL-C) levels increased 66%. Doxazosin did not alter any parameters of blood coagulation measured; however, bleeding times were increased significantly (33%) in doxazosin-treated animals. Although collagen-stimulated platelet aggregation was not influenced by either chronic doxazosin or aspirin treatment, the maximal extent of ADP-stimulated platelet aggregation was significantly reduced (-26% and -18%, respectively) compared with the control monkeys. Platelets from untreated control animals displayed reductions in the extent of ADP-stimulated aggregation of 13% and 23%, respectively, when incubated in vitro with 200 and 300 micrograms/ml of doxazosin. Additionally, the decrease in aggregation response of platelets obtained from doxazosin-treated monkeys was accompanied by a rapid reversal of platelet aggregation. Adhesion to HUVEC by platelets isolated from doxazosin-treated animals was significantly decreased; however, adhesion was not altered when platelets from untreated control animals were incubated with HUVEC in the presence of doxazosin. Thus, the ex vivo and in vitro studies reported in this communication suggest that doxazosin administration to nonhuman primates is associated with beneficial alterations in plasma lipids, platelet aggregation, bleeding times and platelet adhesion to endothelial cells, parameters which are thought to influence risk of cardiovascular disease in both animals and humans.

Animals↗
PubMed1990

Long-range interactions in mammalian platelet aggregation. II. The role of platelet pseudopod number and length.

In part 1, we reported that human (H) platelets, activated with high concentrations (10 microM) of adenosine diphosphate, aggregate under Brownian diffusion (nonstirred, platelet-rich plasma) with an apparent efficiency of collision (alpha B) approximately 4 times and 8 times larger than observed, respectively, for canine (C) and rabbit (R) platelets. Further evaluations of parallel inhibition of alpha B and shape change suggested a central role for platelet pseudopods in mediating the long-range interactions associated with the elevated alpha B values. We found that greater than 90% of all platelet contacts in the doublets and triplets formed were via at least one pseudopod. We therefore compared pseudopod number and length per platelet generated by approximately 30 s post ADP activation in nonstirred PRP from human, canine, and rabbit donors, using phase-contrast, video-enhanced microscopy of fixed platelets. Theoretical calculations assessing the effects of pseudopod length and number on the collision frequency enhanced by an increased radius of a collision sphere supported the experimental observations that approximately 3 or 4 pseudopods per human or canine platelet, and approximately 5 or 6 pseudopods per rabbit platelet yield optimal alpha B values, with the average pseudopod length: approximately 3:2:1 for H/C/R, paralleling the alpha B differences. After correcting for effects of pseudopods and platelet size on platelet diffusion and sedimentation, it still appeared that the small number of long pseudopods formed on human platelets could largely explain the unusually large alpha B values. The quantitative discrepancies between theory and experiment do not appear related to time-dependent refractoriness within the less than 60 s of observation, but may be related to biochemical differences in dynamics and surface density of adhesive (sticky sites) present on the pseudopod surface.

Adenosine Diphosphate↗
PubMed2000

Role of the recombinant protein of the platelet receptor for type I collagen in the release of nitric oxide during platelet aggregation.

Nitric oxide plays an important role in platelet function and platelets possess the endothelial isoform of nitric oxide synthase. Several reports have indicated that nitric oxide is released upon exposure of platelets to collagen. We have reported that a non-integrin platelet protein of 65 kDa is a receptor for type I collagen. By direct measurement of NO release from washed human platelets suspended in Tyrode buffer with a ISO-NO Mark II, World Precision Instruments, Sarasota, FL, USA, p30 sensor, type I collagen, but not ADP and epinephrine, induces the release of NO in a time-dependent manner. The production of NO is inhibited either by preincubation of type I collagen with the platelet type I collagen receptor recombinant protein or by preincubation of platelets with the antibody to the receptor protein, the anti-65 antibody. However, preincubation of platelets with anti-P-selectin and anti-glycoprotein IIb/IIIa did not affect the release of NO by platelets. These results suggest that the 65 kDa platelet receptor for type I collagen is specifically linked to the generation of NO, and that the 65 kDa platelet receptor for type I collagen plays an important new role in platelet function.

Blood Platelets↗
PubMed1999

Platelet aggregation may not be a prerequisite for collagen-stimulated platelet generation of nitric oxide.

By determining the sum of the supernatant concentrations of nitrite and nitrate the stimulated generation of nitric oxide (NO) by human washed platelets induced by a range of fibrillar collagen concentrations (0.0156-25 microg ml(-1)) was investigated. Platelet serotonin (5-hydroxytryptamine, 5-HT) efflux and platelet aggregation were also measured. Under resting conditions (0 microg ml(-1) collagen) platelet NO release was equivalent to 1.06+/-0.17 nmol per 10(8) platelets. Maximal NO release, equivalent to 2.1+/-0. 37 nmol per 10(8) platelets, was observed with only 0.0625 microg ml(-1) collagen (P<0.02, stimulated vs. resting release), higher collagen concentrations producing no further increases in platelet NO output. By contrast, maximal platelet aggregation and 5-HT efflux did not occur until collagen concentrations of 2.5 microg ml(-1) and 10-25 microg ml-1), respectively, had been achieved. L-NAME (1 mmol l(-1)) and L-NMMA (1 mmol l(-1)) inhibited stimulated platelet NO generation by 78+/-6% and 72%, respectively. Contrasting with fibrillar collagen, fibrillar beta-amyloid protein had no effect on platelet NO generation, or on 5-HT efflux or aggregation. These data perhaps indicate that NO generation by human platelets is stimulated by concentrations of fibrillar collagen insufficient to elicit an aggregatory response. Such a mechanism could operate in vivo to inhibit platelet aggregation which might otherwise be induced by low concentrations of circulating agonists.

Blood Platelets↗
PubMed1996

Factor X bound to the surface of activated human platelets is preferentially activated by platelet-bound factor IXa.

Factor X is a zymogen in the blood coagulation system which is activated by the serine protease, factor IXa, in a reaction that is promoted by the presence of stimulated platelets. We have shown previously that platelets possess a binding site for factor IXa, the occupancy of which is correlated with the rate of factor X activation (Ahmad et al., 1989b,c). Similarly, we have described a different binding site on the surface of activated platelets to which the substrate for this reaction, factor X, can bind (see the accompanying paper). This "zymogen binding site" is of moderate affinity and is relatively nonspecific; apparently shared to some degree by factor X and other vitamin K-dependent proteins, most notably prothrombin. We have found that prothrombin fragment 1 not only is able to displace factor X from this platelet binding site but also possesses the ability to inhibit the platelet-dependent activation of factor X. We have developed two mathematical models for the activation of factor X by platelet-bound factor IXa. The first model assumes that factor X is activated in a manner that is totally unrelated to the presumptive zymogen binding site, whereas the second model requires factor X to first bind to this site before it may interact with platelet-bound factor IXa and become activated. Within the context of each of these models, we have evaluated three mechanisms by which prothrombin fragment 1 may inhibit factor X activation. The data presented herein are most consistent with the precept that platelet-bound factor X is activated by platelet-bound factor IXa (kcat approximately 0.0011 s-1) in an explicitly two-dimensional reaction (Km.2D approximately 230 molecules per platelet). Prothrombin fragment 1 is believed to disrupt this reaction by competing with factor X for the zymogen binding site (Ki approximately 470 nM) and, to a lesser degree, by displacing factor IXa from its binding site (Ki approximately 7 microM). These findings suggest that platelet-bound zymogen factor X represents a kinetically important pool of substrate that is preferentially activated on the surface of activated platelets.

Binding Sites↗
PubMed2001

Platelet aggregation is not initiated by platelet shape change.

Because the initial decrease in light transmission in platelet aggregometry is attributed to platelet shape change, it is widely held that platelet shape change is a prerequisite for platelet aggregation. We conducted this study to determine the basis of this initial optical effect in aggregometry. Platelets were activated with ADP, thrombin, or the thrombin receptor agonist peptide SFLLRN (TRAP(1-6)). In every case the initial decrease in light transmission occurred with the concomitant formation of microaggregates. This was also seen when preactivated platelets, which cannot undergo further morphological changes, were used, and when platelets were activated in the presence of shape-change inhibitors such as cytochalasin D and vincristine. Microscopy analysis of samples fixed at minimum light transmission in the aggregometer, which is generally assumed to signal shape change, always showed the presence of microaggregates. Microaggregation appeared to be distinct from full aggregation, as it was not inhibited by the addition of CD61, an antibody to the beta(3) integrin. To model these findings, fibrinogen-coated latex spheres, which cannot change shape, were aggregated with thrombin; the initial decrease in light transmission was still seen, and microaggregates formed at this time. These results indicate that platelet shape change is not a prerequisite for aggregation and that the signal widely believed to represent shape change reflects platelet microaggregation instead. We conclude that platelet aggregation occurs independently of shape change and that shape change is not necessarily followed by aggregation. These observations suggest an alternative role for platelet shape change of single platelets.

Adenosine Diphosphate↗
PubMed1997

Platelet transfusion requirements during autologous peripheral blood progenitor cell transplantation correlate with the pretransplant platelet count.

The use of primed peripheral blood progenitor cells (PBPC) has improved platelet engraftment following autologous bone marrow/PBPC transplantation (ABMT). The thrombocytopenia associated with ABMT generally lasts 14-18 days, and is associated with variable platelet transfusion requirements. Little, if any, data exist examining prognostic parameters for platelet transfusion requirements during autologous transplantation. We retrospectively examined 286 consecutive patients undergoing autologous transplantation from 1 January 1994 to 1 June 1996 with respect to platelet engraftment and platelet transfusion requirements. One hundred and fifty four patients were transplanted for breast cancer (54%), 72 for non-Hodgkin's lymphoma (25%), 35 for Hodgkin's disease (12%), 13 for acute leukemia (5%), eight for myeloma (3%), and four for other malignancy (1%). The median age was 44. All patients received cytokine priming, usually with G-CSF, for the procurement of PBPC. The median number of CD34+ cells collected was 4.3 x 10(6)/kg. All patients received a chemotherapeutic preparative regimen and all received an autologous transplant using PBPC alone. The median time to a platelet count of 20 x 10(9)/l was 13 days. Patients beginning the transplant with a less than normal platelet count (less than 150 x 10(9)/l) engrafted in 17 days, and received a median number of seven platelet transfusions, as compared with platelet engraftment of 12 days, and four platelet transfusions, for patients beginning the transplant with a normal platelet count (P = 0.001). Both groups of patients received an equivalent dose of CD34+ cells. We conclude that thrombocytopenia at the initiation of autologous transplantation is associated with increased platelet transfusion requirements, independent of the dose of CD34+ cells infused.

Adult↗
PubMed1978

Inhibition of adenylate cyclase by adenosine analogues in preparations of broken and intact human platelets. Evidence for the unidirectional control of platelet function by cyclic AMP.

Whereas adenosine itself exerted independent stimulatory and inhibitory effects on the adenylate cyclase activity of a platelet particulate fraction at low and high concentrations respectively, 2-substituted and N6-monosubstituted adenosines had stimulatory but greatly decreased inhibitory effects. Deoxyadenosines, on the other hand, had enhanced inhibitory but no stimulatory effects. The most potent inhibitors found were, in order of increasing activity, 9-(tetrahydro-2-furyl)adenine (SQ 22536), 2',5'-dideoxyadenosine and 2'-deoxyadenosine 3'-monophosphate. Kinetic studies on prostaglandin E1-activated adenylate cyclase showed that the inhibition caused by either 2',5'-dideoxyadenosine or compound SQ 22536 was non-competitive with MgATP and that the former compound, at least, showed negative co-operativity; 50% inhibition was observed with 4 micron-2',5'-dideoxyadenosine or 13 micron-SQ 22536. These two compounds also inhibited both the basal and prostaglandin E1-activated adenylate cyclase activities of intact platelets, when these were measured as the increases in cyclic [3H]AMP in platelets that had been labelled with [3H]adenine and were then incubated briefly with papaverine or papaverine and prostaglandin E1. Both compounds, but particularly 2',5'-dideoxyadenosine, markedly decreased the inhibition by prostaglandin E1 of platelet aggregation induced by ADP or [arginine]vasopressin as well as the associated increases in platelet cyclic AMP, so providing further evidence that the effects of prostaglandin E1 on platelet aggregation are mediated by cyclic AMP. 2'-Deoxyadenosine 3'-monophosphate did not affect the inhibition of aggregation by prostaglandin E1, suggesting that the site of action of deoxyadenosine derivatives on adenylate cyclase is intracellular. Neither 2',5'-dideoxyadenosine nor compound SQ 22536 alone induced platelet aggregation. Moreover, neither compound potentiated platelet aggregation or the platelet release reaction when suboptimal concentrations of ADP, [arginine]vasopressin, collagen or arachidonate were added to heparinized or citrated platelet-rich plasma in the absence of prostaglandin E1. These results show that cyclic AMP plays no significant role in the responses of platelets to aggregating agents in the absence of compounds that increase the platelet cyclic AMP concentration above the resting value.

Adenosine↗
PubMed1995

Mechanisms of platelet activation by a stimulatory antibody: cross-linking of a novel platelet receptor for monoclonal antibody F11 with the Fc gamma RII receptor.

The mechanisms by which a stimulatory monoclonal antibody (mAb), called mAb F11, induces granular secretion and aggregation in human platelets have been characterized. Fab fragments of mAb F11, as well as an mAb directed against the platelet Fc gamma RII receptor (mAb IV.3) were found to inhibit mAb F11-induced platelet secretion and aggregation, indicating that the mAb F11 IgG molecule interacts with the Fc gamma RII receptor through its Fc domain and with its own antigen through its Fab domain. The mAb F11 recognized two platelet proteins of 32 and 35 kDa on the platelet membrane surface, as identified by Western blot analysis. We purified both proteins from human platelet membranes using DEAE-Sepharose chromatography followed by mAb F11 affinity chromatography. When added to platelet-rich plasma, the purified proteins dose-dependently inhibited mAb F11-induced platelet aggregation. The purified protein preparation also competitively inhibited the binding of 125I-labelled mAb F11 to intact platelets. The N-terminal 26 amino acid sequences of both the 32 and 35 kDa proteins were identical and contained a single unblocked serine in the N-terminal position. When digested with N-glycanase, the 32 and 35 kDa proteins were converted into a single approximately 29 kDa protein, indicating that these two proteins are derived from the same core protein but differ in their degree of glycosylation. Internal amino acid sequence analysis of the F11 antigen provided information concerning 68 amino acids and suggested two consensus phosphorylation sites for protein kinase C (PKC). The phosphorylation by PKC of the isolated F11 antigen was observed following stimulation by phorbol 12-myristate 13-acetate. Databank analysis of the N-terminal and internal amino acid sequences of the F11 antigen indicated that the N-terminal sequence exhibited the highest degree of similarity to the variable region of the alpha-chain of human T-cell receptors (TCR). In contrast, the F11 internal sequences did not exhibit any similarity to the TCR. Our results demonstrate that the F11 antigen is a novel platelet membrane surface glycoprotein which becomes cross-linked with the Fc gamma RII receptor when platelets are activated by the stimulatory mAb F11. These mechanisms may be relevant to the production of immune thrombocytopenia by platelet-activating antibodies.

Amino Acid Sequence↗
PubMed1987

Platelet catecholamines and platelet function in normal human subjects.

We have used high-performance liquid chromatography with electrochemical detection to measure plasma and platelet catecholamines in 24 normal subjects. In the same subjects platelet function was assessed by measuring platelet aggregation in response to adenosine 5'-pyrophosphate, thrombin, adrenaline and collagen. Platelet sensitivity to prostacyclin was also examined. Platelet noradrenaline showed a positive correlation with extent of aggregation induced by 'low-dose' collagen (1 microgram/ml). No correlation was seen at the higher collagen concentration. Platelet noradrenaline content also correlated with sensitivity of platelets to prostacyclin. High platelet noradrenaline concentrations appeared to result in decreased sensitivity to prostacyclin. No other correlations were observed. These data suggest that platelet noradrenaline rather than plasma levels may be involved in modifying platelet function in vivo. Local release of platelet catecholamines may affect the platelet/vessel wall interaction, the primary physiological step in platelet activation.

Adult↗
PubMed1994

Increased platelet volume and platelet mass in patients with atherosclerotic renal artery stenosis.

1. Platelet volume was measured in citrated blood in two groups of patients at risk of having atherosclerotic renal artery stenosis, namely (i) 30 patients with severe hypertension, and (ii) 44 patients with peripheral vascular disease. 2. Platelet volume was increased in patients with hypertension who had atherosclerotic renal artery stenosis diagnosed by angiography: no renal artery stenosis, median 7.2 (interquartile range 0.5) x 10(-15)/l; renal artery stenosis 7.8 (0.8) x 10(-15)/l; platelet mass also increased with increasing severity of renal artery stenosis. Platelet volume correlated with severity of renal artery stenosis (rs = 0.391, 2p = 0.033, n = 30). Similarly, platelet volume correlated with severity of renal artery stenosis in patients with peripheral vascular disease (rs = 0.319, 2p = 0.035, n = 44). Serum immunoreactive platelet-derived growth factor (predominantly released from platelets) and plasma immunoreactive interleukin-6 (a cytokine which has been postulated to regulate platelet volume) concentrations were not different between hypertensive patients with and without renal artery stenosis. 3. Since large platelets are hyperactive, increased platelet volume may contribute to the development of atherosclerotic renal artery stenosis. However, the present data do not lend support to the hypothesis that platelet-derived growth factor is central to renal artery atherogenesis. Interleukin-6 does not appear to regulate platelet volume.

Aged↗
PubMed2002

The effects of platelet apheresis in total hip replacement surgery on platelet activation.

BACKGROUND: Autologous platelet rich plasma (PRP) harvest with autotransfusion devices has been used for 10 years in cardiac surgery and recently in orthopedics as a blood saving method. The quality of the harvested platelets has not been adequately examined, in part because of methodological difficulties in studying platelet function during surgery. METHODS: Twenty patients undergoing primary total hip replacement (THR) were studied. Ten patients underwent an immediate preoperative platelet apheresis to obtain concentrated platelet rich plasma (c-PRP). The other 10 patients not undergoing apheresis were allocated to a control group. Platelet activation was evaluated as the population expressing P-selectin on the surface of platelets in the c-PRP and in blood samples collected pre-, per- and postoperatively. The method used was flow cytometry. RESULTS AND CONCLUSIONS: A minor population of activated platelets was found to be circulating in the patients' blood, with a highly significant difference between patients (P = 0.005), and with a range of 1-23% in peroperative activation. PRP harvest did not significantly alter platelet activity. The platelet apheresis procedure did not inhibit platelet function in the c-PRP, as judged by a high proportion of platelets that could be activated in ADP stimulation experiments (mean value +/- SD 86% +/- 7.5%).

Adenosine Diphosphate↗