Immunological Evidence for Prothrombin in Human Platelets

1986 ◽  
Vol 55 (02) ◽  
pp. 268-270
Author(s):  
R J Alexander
Keyword(s):  
Electrophoretic Mobility ◽  
Platelet Activation ◽  
Double Diffusion ◽  
Human Platelets ◽  
Secreted Proteins ◽  
Platelet Surface ◽  
Activated Platelets ◽  
Similar Protein ◽  
Diffusion Assay ◽  
Supernatant Solution

SummaryAn attempt was made to isolate from plasma the platelet surface substrate for thrombin, glycoprotein V (GPV), because a GPV antigen was reported to be present in plasma (3). Plasma fractionation based on procedures for purification of GPV from platelets revealed a thrombin-sensitive protein with appropriate electrophoretic mobility. The protein was purified; an antiserum against it i) reacted with detergent-solubilized platelet proteins or secreted proteins in a double diffusion assay, ii) adsorbed a protein from the supernatant solution of activated platelets, and iii) inhibited thrombin-induced platelet activation, but the antiserum did not adsorb labeled GPV. The purified protein was immunochemically related to prothrombin rather than to GPV. Other antibodies against prothrombin were also able to adsorb a protein from platelets. It is concluded that 1) plasma does not contain appreciable amounts of GPV, and 2) platelets contain prothrombin or an immunochemically similar protein.

scholarly journals Prostaglandin E2 potentiates platelet aggregation by priming protein kinase C

Blood ◽  
1993 ◽  
Vol 82 (9) ◽  
pp. 2704-2713 ◽  
Author(s):  
R Vezza ◽  
R Roberti ◽  
GG Nenci ◽  
P Gresele
Keyword(s):  
Protein Kinase C ◽  
Platelet Aggregation ◽  
Protein Kinase ◽  
Prostaglandin E2 ◽  
Platelet Activation ◽  
Human Platelets ◽  
Platelet Surface ◽  
Kinase C ◽  
Activated Platelets ◽  
Induced Aggregation

Abstract Prostaglandin E2 (PGE2) is produced by activated platelets and by several other cells, including capillary endothelial cells. PGE2 exerts a dual effect on platelet aggregation: inhibitory, at high, supraphysiologic concentrations, and potentiating, at low concentrations. No information exists on the biochemical mechanisms through which PGE2 exerts its proaggregatory effect on human platelets. We have evaluated the activity of PGE2 on human platelets and have analyzed the second messenger pathways involved. PGE2 (5 to 500 nmol/L) significantly enhanced aggregation induced by subthreshold concentrations of U46619, thrombin, adenosine diphosphate (ADP), and phorbol 12-myristate 13-acetate (PMA) without simultaneously increasing calcium transients. At a high concentration (50 mumol/L), PGE2 inhibited both aggregation and calcium movements. PGE2 (5 to 500 nmol/L) significantly enhanced secretion of beta-thromboglobulin (beta TG) and adenosine triphosphate from U46619- and ADP-stimulated platelets, but it did not affect platelet shape change. PGE2 also increased the binding of radiolabeled fibrinogen to the platelet surface and increased the phosphorylation of the 47-kD protein in 32P- labeled platelets stimulated with subthreshold doses of U46619. Finally, the amplification of U46619-induced aggregation by PGE2 (500 nmol/L) was abolished by four different protein kinase C (PKC) inhibitors (calphostin C, staurosporine, H7, and TMB8). Our results suggest that PGE2 exerts its facilitating activity on agonist-induced platelet activation by priming PKC to activation by other agonists. PGE2 potentiates platelet activation at concentrations produced by activated platelets and may thus be of pathophysiologic relevance.

HMGB1 binds to activated platelets via the receptor for advanced glycation end products and is present in platelet rich human coronary artery thrombi

2015 ◽  
Vol 114 (11) ◽  
pp. 994-1003 ◽  
Author(s):  
Michael Bode ◽  
David Haenel ◽  
Christoph E. Hagemeyer ◽  
Hannah Seeba ◽  
Daniel Duerschmied ◽  
...  
Keyword(s):  
Coronary Artery ◽  
Platelet Activation ◽  
Knockout Mice ◽  
Surface Expression ◽  
Human Platelets ◽  
Wild Type ◽  
Human Coronary Artery ◽  
Specific Expression ◽  
Platelet Surface ◽  
Activated Platelets

SummaryHigh mobility group box 1 (HMGB1) acts as both a nuclear protein that regulates gene expression, as well as a pro-inflammatory alarmin that is released from necrotic or activated cells. Recently, HMGB1-expression in human atherosclerotic plaques was identified. Therapeutic blockade of HMGB1 reduced the development of diet-induced atherosclerosis in ApoE knockout mice. Thus, we hypothesised an interaction between HMGB1 and activated platelets. Binding of recombinant HMGB1 to platelets was assessed by flow cytometry. HMGB1 bound to thrombin-activated human platelets (MFI 2.49 vs 25.01, p=0.0079). Blood from wild-type, TLR4 and RAGE knockout mice was used to determine potential HMGB1 receptors on platelets. HMGB1 bound to platelets from wild type C57Bl6 (MFI 2.64 vs 20.3, p< 0.05), and TLR4-/- mice (MFI 2.11 vs 25.65, p< 0.05) but failed to show binding to platelets from RAGE-/- mice (p > 0.05). RAGE expression on human platelets was detected by RT-PCR with mRNA extracted from highly purified platelets and confirmed by Western blot and immunofluorescence microscopy. Platelet activation increased RAGE surface expression (MFI 4.85 vs 6.74, p< 0.05). Expression of HMGB1 in human coronary artery thrombi was demonstrated by immunohistochemistry and revealed high expression levels. Platelets bind HMGB1 upon thrombin-induced activation. Platelet specific expression of RAGE could be detected at the mRNA and protein level and is involved in the binding of HMGB1. Furthermore, platelet activation up-regulates platelet surface expression of RAGE. HMGB1 is highly expressed in platelet-rich human coronary artery thrombi pointing towards a central role for HMGB1 in atherothrombosis, thereby suggesting the possibility of platelet targeted anti-inflammatory therapies for atherothrombosis.

scholarly journals Prostaglandin E2 potentiates platelet aggregation by priming protein kinase C

Blood ◽  
1993 ◽  
Vol 82 (9) ◽  
pp. 2704-2713
Author(s):  
R Vezza ◽  
R Roberti ◽  
GG Nenci ◽  
P Gresele
Keyword(s):  
Protein Kinase C ◽  
Platelet Aggregation ◽  
Protein Kinase ◽  
Prostaglandin E2 ◽  
Platelet Activation ◽  
Human Platelets ◽  
Platelet Surface ◽  
Kinase C ◽  
Activated Platelets ◽  
Induced Aggregation

Prostaglandin E2 (PGE2) is produced by activated platelets and by several other cells, including capillary endothelial cells. PGE2 exerts a dual effect on platelet aggregation: inhibitory, at high, supraphysiologic concentrations, and potentiating, at low concentrations. No information exists on the biochemical mechanisms through which PGE2 exerts its proaggregatory effect on human platelets. We have evaluated the activity of PGE2 on human platelets and have analyzed the second messenger pathways involved. PGE2 (5 to 500 nmol/L) significantly enhanced aggregation induced by subthreshold concentrations of U46619, thrombin, adenosine diphosphate (ADP), and phorbol 12-myristate 13-acetate (PMA) without simultaneously increasing calcium transients. At a high concentration (50 mumol/L), PGE2 inhibited both aggregation and calcium movements. PGE2 (5 to 500 nmol/L) significantly enhanced secretion of beta-thromboglobulin (beta TG) and adenosine triphosphate from U46619- and ADP-stimulated platelets, but it did not affect platelet shape change. PGE2 also increased the binding of radiolabeled fibrinogen to the platelet surface and increased the phosphorylation of the 47-kD protein in 32P- labeled platelets stimulated with subthreshold doses of U46619. Finally, the amplification of U46619-induced aggregation by PGE2 (500 nmol/L) was abolished by four different protein kinase C (PKC) inhibitors (calphostin C, staurosporine, H7, and TMB8). Our results suggest that PGE2 exerts its facilitating activity on agonist-induced platelet activation by priming PKC to activation by other agonists. PGE2 potentiates platelet activation at concentrations produced by activated platelets and may thus be of pathophysiologic relevance.

scholarly journals Factor XIIIa binding to activated platelets is mediated through activation of glycoprotein IIb-IIIa

Blood ◽  
1994 ◽  
Vol 83 (4) ◽  
pp. 1006-1016 ◽  
Author(s):  
AD Cox ◽  
DV Devine
Keyword(s):  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
Platelet Activation ◽  
Factor Xiiia ◽  
Cross Linking ◽  
Platelet Surface ◽  
Activated Platelets ◽  
Fibrinogen Binding ◽  
A Chain ◽  
Functional Receptor

Abstract Stabilization of a clot is dependent on fibrin cross-linking mediated by the transglutaminase, factor XIIIa (FXIIIa). In addition to fibrin stabilization, FXIIIa acts on a number of platelet-reactive proteins, including fibronectin and vitronectin, as well as the platelet proteins, glycoprotein (GP) IIb-IIIa, myosin, and actin. However, conditions inducing the platelet-activation dependent binding of FXIIIa have not been characterized nor have the sites mediating FXIIIa binding been identified. The generation of FXIIIa and consequent detection of FXIIIa on the platelet surface were compared with other thrombin- induced activation events; the rate at which FXIIIa bound to activated platelets was much slower than platelet degranulation or fibrin(ogen) binding. Whereas platelets could be rapidly induced to express a functional receptor for FXIIIa, the rate of FXIIIa binding to platelets is limited by the rate of conversion of FXIII to FXIIIa. Immunoprecipitation of radiolabeled platelets using polyclonal anti- FXIII A-chain antibody identified two proteins corresponding to GPIIb and GPIIIa. Preincubation of intact platelets with 7E3, a monoclonal antibody that blocks the fibrinogen binding site, or GRGDSP peptide inhibited FXIIIa binding by about 95% when measured by flow cytometry; FXIIIa binding to purified GPIIb-IIIa was also inhibited by 7E3. The binding of FXIIIa to purified GPIIb-IIIa was enhanced by the addition of fibrinogen, but not by that of fibronectin or thrombospondin, suggesting that FXIIIa also binds to fibrinogen associated with the complex. These observations suggest that activated platelets bearing FXIIIa may enhance stabilization of platelet-rich thrombi through surface-localized cross-linking events.

DETECTION OF ACTIVATED PLATELETS IN WHOLE BLOOD BY FLOW CYTOMETRY

1987 ◽  
Author(s):  
S J Shattil ◽  
J A Hoxie ◽  
M Cunningham ◽  
C S Abrahms ◽  
J O’Brien ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Monoclonal Antibodies ◽  
Platelet Activation ◽  
Whole Blood ◽  
Thrombus Formation ◽  
Membrane Surface ◽  
White Blood Cells ◽  
Color Flow ◽  
Platelet Surface ◽  
Activated Platelets

Platelets may become activated in a number of clinical disorders and participate in thrombus formation. We have developed a direct test for activated platelets in whole blood that utilizes dual-color flow cytometry and requires no washing steps. Platelets were distinguished from erythrocytes and white blood cells in the flow cytometer by labeling the platelets with biotin-AP1, an antibody specific for membrane glycoprotein lb, and analyzing the cells for phycoerythrin-streptavidin fluorescence. Membrane surface changes resulting from platelet activation were detected with three different FITC-labeled monoclonal antibodies: 1) PAC1, an antibody specific for the fibrinogen receptor on activated platelets; 2) 9F9, which binds to the D-domain of fibrinogen and detects platelet-bound fibrinogen; and 3) S12, which binds to an alpha-granule membrane protein that associates with the platelet surface during secretion. Unstimulated platelets demonstrated no PAC1, 9F9, or S12-specific fluorescence, indicating that they did not bind these antibodies. Upon stimulation with agonists, however, the platelets demonstrated a dose-dependent increase in FITC-fluorescence. The binding of 9F9 to activated platelets required fibrinogen. Low concentrations of ADP and epinephrine, which induce fibrinogen receptors but little secretion, stimulated near-maximal PAC1 or 9F9 binding but little S12 binding. On the other hand, a concentration of phorbol myristate acetate that evokes full platelet aggregation and secretion induced maximal binding of all three antibodies. When blood samples containing activated and non-activated platelets were mixed, as few as 0.8% activated platelets could be detected by this technique. There was a direct correlation between ADP-induced FITC-PAC1 binding and binding determined in a conventional 125I-PAC1 binding assay (r = 0.99; p < 0.001). These studies demonstrate that activated platelets can be reliably detected in whole blood using activation-dependent monoclonal antibodies and flow cytometry. This method may be useful to assess the degree of platelet activation and the efficacy platelet inhibitor therapy in thrombotic disorders.

Regulated Shedding of sema4D from the Platelet Surface Produces a Bioactive Second Messenger in Thrombotic Disorders.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1641-1641
Author(s):  
Li Zhu ◽  
Wolfgang Bergmeier ◽  
Jie Wu ◽  
Hong Jiang ◽  
Nana Yeboah ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Platelet Activation ◽  
Transmembrane Domain ◽  
Thrombus Formation ◽  
Active Form ◽  
Extracellular Domain ◽  
Human Platelets ◽  
Biologically Active ◽  
Western Blots ◽  
Platelet Surface

Abstract Proteins that are expressed on the platelet surface can participate in contact-dependent signaling events which modulate thrombus formation or, after being shed from the platelet surface, serve as bioactive messengers that affect the function of nearby cells. Here we show for the first time that platelets express the class IV semaphorin known as sema4D or CD100, and that platelet activation causes the regulated shedding of the sema4D extracellular domain in a biologically-active form. Sema4D is a glycosylated 150 kDa disulfide-linked homodimer that has previously been implicated in interactions between T-cells and B-cells. Platelet activation by collagen, thrombin or PMA causes a transient increase in sema4D surface expression peaking at 15 min, followed by a complete loss of expression over 30–60 minutes. These events are accompanied by the release of the sema4D exodomain as a 130 kDa fragment, leaving a 25–30 kDa transmembrane and cytoplasmic domain fragment that is retained by the platelets. The cleavage event required to produce these fragments is inhibited by metalloprotease inhibitors and abolished in platelets from chimeric mice lacking the metalloprotease known as ADAM17 or TACE (TNF alpha converting enzyme). Incubation of recombinant sema4D with ADAM17 identified a single cleavage site just outside the predicted transmembrane domain. Western blots show that human platelets express ADAM17 in both its immature (zymogen) and mature (active) forms, and indicate that at least some of the ADAM17 is located on the platelet surface. ADAM17-dependent cleavage of sema4D does not require platelet aggregation, but the rate is accelerated when aggregation is allowed to occur and slowed when aggregation is prevented. Under both sets of conditions, cleavage of sema4D occurs to a greater extent and more rapidly than the ADAM17-dependent cleavage of GP Ib alpha, suggesting that there is a hierarchy of proteolytic events when platelets are activated. In terms of biological impact, the shedding of sema4D following platelet activation raises the possibility that the soluble extracellular domain of sema4D serves as a bioactive messenger. Two receptors for sema4D have been identified previously: CD72, which is present on lymphocytes where it regulates the activity of the tyrosine phosphatase, SHP-1, and plexin-B1, which is expressed in endothelial cells. Western blots suggest that both of these receptors are expressed on human platelets and show that SHP-1 is associated with CD72 in resting, but not activated platelets. Taken together, these results demonstrate that sema4D undergoes regulated ADAM17-dependent shedding when platelets are activated, and suggest that this results in the production of a bioactive form of the molecule that can affect responses in nearby platelets, lymphocytes and endothelial cells at sites of thrombosis.

Junctional Adhesion Molecule a Helps Maintain Integrin αIIbβ3 in Resting State

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 111-111 ◽  
Author(s):  
Meghna Ulhas Naik ◽  
Timothy J. Stalker ◽  
Lawrence F. Brass ◽  
Ulhas Pandurang Naik
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Adhesion Molecule ◽  
Resting State ◽  
Human Platelets ◽  
Artery Occlusion ◽  
In Vivo Model ◽  
Platelet Surface ◽  
Integrin Αiibβ3

Abstract Under physiological conditions, fibrinogen receptor integrin αIIbβ3 on the circulating platelets is in a low-affinity, or resting state, unable to bind soluble ligands. During platelet activation by agonists, a cascade of signaling events induces a conformational change in the extracellular domain of αIIbβ3, thereby converting it into a high-affinity state capable of binding ligands through a process known as “inside-out signaling”. What maintains this integrin in a low-affinity state is not well understood. We have previously identified JAM-A, junctional adhesion molecule A, on the platelet surface. We have shown that an antibody blockade of JAM-A dose-dependently activates platelets. To understand the molecular mechanism through which JAM-A regulates platelet aggregation, we used Jam-A null mice. Interestingly, the mouse bleeding times were significantly shortened in Jam-A null mice compared to wildtype littermates. Furthermore, the majority of these mice showed a rebleeding phenotype. This phenotype was further confirmed by FeCl3-induced carotid artery occlusion, a well-accepted in vivo model for thrombosis. Platelets derived from Jam-A-null mice were used to evaluate the role of JAM-A in agonist-induced platelet aggregation. We found that Jam-A null platelets showed enhanced aggregation in response to physiological agonists such as PAR4 peptide, collagen, and ADP as compared to platelets from wildtype littermates. JAM-A was found to associate with αIIbβ3 in unactivated human platelets, but this association was disrupted by both agonist-induced platelet aggregation and during outside-in signaling initiated upon platelet spreading on immobilized Fg. We also found that in resting platelets, JAM-A is phosphorylated on a conserved tyrosine 280 in its cytoplasmic domain, which was dephosphorylated upon platelet activation. Furthermore, JAM-A is rapidly and transiently phosphorylated on serine 284 residue during platelet activation by agonists. Interestingly, JAM-A also formed a complex with Csk, a tyrosine kinase known to be inhibitory to Src activation, in resting platelets. This complex was dissociated upon activation of platelets by agonists. These results suggest that tyrosine-phosphorylated JAM-A recruits Csk to αIIbβ3 in resting platelets, thus maintaining a low-affinity state of integrin αIIbβ3. Agonist–induced activation of platelets results in rapid dephosphorylation of JAM-A on Y280 and phosphorylation on S284 residues. This causes dissociation of JAM-A from integrin αIIbβ3 facilitating platelet aggregation.

Functional expression of CCR1, CCR3, CCR4, and CXCR4 chemokine receptors on human platelets

Blood ◽  
2000 ◽  
Vol 96 (13) ◽  
pp. 4046-4054
Author(s):  
Kenneth J. Clemetson ◽  
Jeannine M. Clemetson ◽  
Amanda E. I. Proudfoot ◽  
Christine A. Power ◽  
Marco Baggiolini ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Messenger Rna ◽  
Chemokine Receptors ◽  
Polyclonal Antibodies ◽  
Adenosine Diphosphate ◽  
Functional Expression ◽  
Human Platelets ◽  
Platelet Factor ◽  
Platelet Surface ◽  
Stromal Cell Derived Factor

Platelets are known to contain platelet factor 4 and β-thromboglobulin, α-chemokines containing the CXC motif, but recent studies extended the range to the β-family characterized by the CC motif, including RANTES and Gro-α. There is also evidence for expression of chemokine receptors CCR4 and CXCR4 in platelets. This study shows that platelets have functional CCR1, CCR3, CCR4, and CXCR4 chemokine receptors. Polymerase chain reaction detected chemokine receptor messenger RNA in platelet RNA. CCR1, CCR3, and especially CCR4 gave strong signals; CXCR1 and CXCR4 were weakly positive. Flow cytometry with specific antibodies showed the presence of a clear signal for CXCR4 and weak signals for CCR1 and CCR3, whereas CXCR1, CXCR2, CXCR3, and CCR5 were all negative. Immunoprecipitation and Western blotting with polyclonal antibodies to cytoplasmic peptides clearly showed the presence of CCR1 and CCR4 in platelets in amounts comparable to monocytes and CCR4 transfected cells, respectively. Chemokines specific for these receptors, including monocyte chemotactic protein 1, macrophage inflammatory peptide 1α, eotaxin, RANTES, TARC, macrophage-derived chemokine, and stromal cell–derived factor 1, activate platelets to give Ca++ signals, aggregation, and release of granule contents. Platelet aggregation was dependent on release of adenosine diphosphate (ADP) and its interaction with platelet ADP receptors. Part, but not all, of the Ca++ signal was due to ADP release feeding back to its receptors. Platelet activation also involved heparan or chondroitin sulfate associated with the platelet surface and was inhibited by cleavage of these glycosaminoglycans or by heparin or low molecular weight heparin. These platelet receptors may be involved in inflammatory or allergic responses or in platelet activation in human immunodeficiency virus infection.

Occurrence of Amphoterin (HMG1) as an Endogenous Protein of Human Platelets that Is Exported to the Cell Surface upon Platelet Activation

2000 ◽  
Vol 84 (12) ◽  
pp. 1087-1094 ◽  
Author(s):  
Shinji Imai ◽  
Heikki Rauvala ◽  
Jaakko Parkkinen ◽  
Ari Rouhiainen
Keyword(s):  
Cell Surface ◽  
Platelet Activation ◽  
Divalent Cations ◽  
Leading Edge ◽  
Human Platelets ◽  
Lipid Binding ◽  
Cell Surface Receptor ◽  
Heparin Binding ◽  
Platelet Surface ◽  
Endogenous Protein

SummaryAmphoterin (HMG1) is a 30-kD heparin-binding protein which is functionally associated with the outgrowth of cytoplasmic processes in developing neurones. Amphoterin has been shown to mediate adhesive and proteolytic interactions at the leading edge of motile cells. Recently it was shown that inhibition of amphoterin interactions with its cell surface receptor (RAGE) suppresses tumour growth and metastasis. In this work we have identified amphoterin polypeptide and its mRNA in human platelets. Amphoterin had a cytoplasmic localisation in resting platelets according to subcellular fractionation studies and immunogold electronmicroscopy. After platelet activation, part of amphoterin was associated with the external surface of plasma membrane. Externalisation of amphoterin during platelet activation was also detected in immunofluorescence studies. Amphoterin was detectable in human serum (0.2 ng/ml) but not in plasma. Resting platelets treated with PGI2 and forskolin bound to immobilised recombinant amphoterin independently of divalent cations. The binding induced a spicular morphology in platelets, and was effectively inhibited by heparin. Amphoterinbinding protein components on the platelet surface were not identified, but amphoterin bound to phosphatidylserine and sulfatide in lipid binding assays. Our results suggest that amphoterin is an endogenous protein in human platelets, which is exported to the cell surface during platelet activation. Interaction of amphoterin with the platelet surface may be mediated by sulfoglycolipids and phospholipids.

scholarly journals Direct detection of activated platelets and platelet-derived microparticles in humans

Blood ◽  
1990 ◽  
Vol 75 (1) ◽  
pp. 128-138 ◽  
Author(s):  
CS Abrams ◽  
N Ellison ◽  
AZ Budzynski ◽  
SJ Shattil
Keyword(s):  
Flow Cytometry ◽  
Cardiopulmonary Bypass ◽  
Platelet Activation ◽  
Bleeding Time ◽  
Relative Proportion ◽  
Open Heart Surgery ◽  
Normal Range ◽  
In Vivo Models ◽  
Platelet Surface ◽  
Activated Platelets

Flow cytometry was used to determine whether activated platelets and platelet-derived microparticles can be detected directly in whole blood after a hemostatic insult. Two different in vivo models of platelet activation were examined: (1) a standardized bleeding time, and (2) cardiopulmonary bypass. Platelets and microplatelets were identified with a biotinylated anti-glycoprotein (GP)lb antibody and a fluorophore, phycoerythrin-streptavidin. Microparticles were distinguished from platelets by light scatter. Activated platelets were detected with three fluorescein-labeled monoclonal antibodies (MoAbs): (1) PAC1, which binds to the activated form of GPIIb-IIIa; (2) 9F9, a newly developed antibody that is specific for fibrinogen bound to the surface of activated platelets; and (3) S12, which binds to an alpha- granule membrane protein expressed on the platelet surface after granule secretion. In nine normal subjects, bleeding times ranged from 4.5 to 7.5 minutes. Over this time, there was a progressive increase in the amount of PAC1, 9F9, and S12 bound to platelets in blood emerging from the bleeding time wound. With all three antibodies, platelet activation was apparent as early as 30 seconds after the incision (P less than .03). Activation was accompanied by a progressive decrease in the concentration of platelets in blood from the wound, while the concentration of microparticles increased slightly. In nine patients undergoing open heart surgery, 1 hour of cardiopulmonary bypass caused a 2.2-fold increase in the relative proportion of microparticles in circulating blood (P less than .001). Moreover, bypass caused platelet activation as evidenced by a mean two- to threefold increase in PAC1 binding to platelets. Although this increase was significant (P less than .02), PAC1 binding exceeded the normal range for unstimulated control platelets in only 5 of 9 patients, and 9F9 and S12 binding exceeded the normal range in only two patients. Taken together, these studies demonstrate that it is now feasible using flow cytometry to evaluate the extent of platelet activation and the presence of platelet- derived microparticles in the circulation of humans.

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