Washed Platelet System Research Articles

Procoagulant Platelet Subpopulation Formation in Whole Blood: Correlation with Whole Blood Clotting Time and 5-Year Mean Bleeding Score in Individuals with Factor VIII Deficiency.

Abstract 4022Poster Board III-958Different clinical phenotypes are often observed among individuals with hemorrhagic or thrombotic disorders despite the presence of the same mutation and/or factor levels suggesting that additional genetic and/or environmental factors influence clinical presentation. As thrombin plays a central role in hemostasis, factors that affect an individual's ability to generate thrombin may lead to an increased risk of hemorrhage or thrombosis. The goal of this study was to assess procoagulant platelet formation in individuals with hemophilia A using a rapid, whole blood flow cytometric assay of prothrombinase complex assembly on platelets described previously. In this assay, Ca2+-dependent factor Xa binding to activated platelets is used as a marker of thrombin generating potential as it has been shown previously to correlate with platelet prothrombinase activity in a washed platelet system. Procoagulant platelet subpopulation formation in 15 men with varying degrees of factor VIII-deficiency was evaluated and compared to two independent measures of hemostatic competence: a 5-year mean bleeding score and whole blood clot time. In these individuals, the % activated platelets binding factor Xa in whole blood varied from 2.35 – 9.0% (n = 30), which is consistent with what is observed in unaffected individuals (1.5 – 41.5%, n = 136). Bleeding was scored independently by two experienced hemophilia nurses and one hematologist at Centre Hospitalier Universitaire Sainte Justine based on each individual's bleeding history, including hemarthroses, soft tissue hematoma, and annual factor VIII usage, and averaged. The 5-year mean bleeding scores in these individuals ranged from 0 – 20.4. Linear regression analyses indicated that in this population the % activated platelets binding factor Xa correlated indirectly with the 5-year mean bleeding score, where individuals with lower bleeding scores (i.e. less bleeding episodes and/or factor VIII usage) generated larger procoagulant platelet subpopulations. The time to clot formation in a tissue factor-dependent, contact pathway-suppressed whole blood clotting assay described previously, was also determined. The clot time, which was determined visually, and ranged from ∼3 – 7 min, also correlated indirectly, though less well, with the % activated platelets binding factor Xa in whole blood (r = -0.23). Thus, consistent with what was observed with the bleeding score, those individuals who clotted more quickly (i.e. exhibited a greater degree of hemostatic competence) generated more proacoagulant platelets. Platelet procoagulant subpopulation formation was also compared to other hematological measures. An inverse correlation was observed between the % activated platelets binding factor Xa and mean platelet volume (r = -0.347), suggesting that in this population, individuals with smaller platelets (i.e. platelets with fewer prothrombinase binding sites) may generate a greater number of platelets capable of assembling prothrombinase. In contrast, no correlation was observed between whole blood platelet number and % activated platelets binding factor Xa. Interestingly, factor Xa binding was also negatively correlated with plasma levels of factor IX (r = -0.56) and factor V (r = -0.62). Platelet subpopulation formation was also weakly and negatively associated with the aPTT (r = - 0.28). In this small population of individuals with hemophilia A, whole blood platelet factor Xa binding correlates with bleeding phenotype. These observations support the notion that this measurement can be used to predict an individuals propensity to bleed or clot. Disclosures:No relevant conflicts of interest to declare.

Platelet Sensitivity is Increased by Acrolein

Acrolein is a toxic environmental aldehyde. It is also produced during lipid peroxidation. Since acrolein exposure is associated with cardiovascular distress, we studied its effect on mouse platelets. In a platelet aggregation assay, acrolein (10–25μM) added to platelets‐rich plasma (PRP) or fibrinogen (fg) (200nM) added washed platelets, 30s prior to 10μM ADP or 250μM arachidonic acid, significantly augmented aggregation (15–20%) when compared with untreated platelets. To study in vivo effects, blood from mice fed with 1–5 mg/kg/day acrolein for 1–3 days was pooled. ADP‐induced platelet aggregation was augmented (10–20%) in a dose and time dependent manner in acrolein treated animals, both in the PRP and washed platelet system. In acrolein treated mice, plasma levels of platelet factor‐4 and P‐selectin were significantly increased, platelet‐monocyte aggregates in blood increased by 15% and the tail bleeding time was shortened by 25% while plasma fg levels were unchanged. Mice exposed to acrolein inhalation for 6h also showed augmented ADP‐induced platelet aggregation. These data suggest that acrolein exposure at environmentally relevant concentrations could increase platelet adhesion in vivo and such effects may be responsible in part for the increased thrombogenicity of acrolein containing pollutants such as automobile exhaust and cigarette smoke. The work was funded by NIEHS grant ES11860.

Mechanisms of Platelet Activation by Antithymocyte Globulin (ATG).

We have previously shown that polyclonal rabbit ATG from Fresenius (ATG-F) can induce non-overt DIC in patients undergoing hematopoietic stem cell transplantation (HSCT) [Weber et al. 2003]. The compensated state of the coagulopathy suggested that the severe drop in platelet counts (>60%) observed in patients with normal pretreatment levels was not solely due to thrombin generation and platelet consumption. We thus further investigated the mechanisms of ATG-F-induced thrombocytopenia, hypothesizing that ATG-F had direct platelet-stimulating activity. Using an indirect flow cytometric binding assay we found that, compared to control IgG, ATG-F dose-dependently (1–100 μg/ml) bound to the surface of resting and TRAP-activated platelets, showing an up to 50fold increase in MFI at 50–100 μg/ml. In a washed platelet system, in which baseline positivity for CD62P was generally <15%, ATG-F alone had no effect on platelet aggregation. However, when platelets were primed with low concentrations of ADP (1–2 μM) or epinephrine (0.5 μM), ATG-F induced strong and stable aggregation of up to 80–100% in a dose-dependent manner (25–100 μg/ml), whereas control IgG did not. The priming effect of ADP was dependent on both P2Y1 and P2Y12 as evidenced by respective ADP receptor antagonists. Preincubation of platelets with inhibitory CD32 mAb completely abolished ATG-F-induced platelet aggregation, suggesting that clustering of, and signalling through, FcγRIIA was crucial for this platelet response. Similarly, ATG-F-derived F(ab′)2 had no effect on ADP-triggered platelet aggregation. Furthermore, preliminary studies indicated that responsiveness of platelet donors to ATG-F was associated with the FcγRIIA-R/R131 polymorphism, which has previously been shown to confer increased in vitro platelet responsiveness to heparin/PF4 antibodies from patients with HIT. In a 14C-serotonin release assay, in which prolonged incubation at 370C under shaking conditions increases baseline platelet activation and ensures constant platelet-platelet contacts, ATG-F (100 μg/ml), but not control IgG, induced strong FcγRIIA-dependent dense-granule release of up to 80%. In contrast, using diluted platelet-rich plasma under static conditions, ATG-F had only minor effects on platelet activation, increasing CD62P+ platelets from 2±1 to 15±5% (n=5). In a cohort of 16 consecutive HSCT patients receiving ATG-F, two patients were identified with normal pretreatment platelet counts and soluble CD62P (sCD62P) plasma levels of >51 ng/ml (mean+2SD of 23 controls). In these patients, an ATG-F-induced drop in platelet count to 38 and 19% of baseline was associated with a 1.7 and 2.2fold increase in sCD62P, respectively, indicating further platelet activation. In summary, strong binding of ATG-F to resting platelets may accelerate their clearance by the reticulo-endothelial system, thus contributing to the pathophysiology of thrombocytopenia. While ATG-F alone had negligible effects on resting platelets, it significantly enhanced activation of prestimulated platelets. Patients with normal-to-high platelet counts and evidence of in vivo platelet activation may especially be prone to this potentially hazardous side effect.

Targeting Angiogenesis in Cancer: Bevacizumab-Induced Platelet Activation as a Possible Cause for Unexpected Arterial Thromboembolic Events in Clinical Trials.

The humanized monoclonal VEGF antibody, bevacizumab (Avastin, Genentech), is approved in combination with standard chemotherapy for first-line treatment of patients with metastatic colorectal cancer (CRC) and also shows promising efficacy as anti-angiogenic immunotherapy in patients with non-small-cell lung cancer (NSCLC). A pooled analysis of five randomized, controlled trials (n=1745) showed that, compared to placebo, bevacizumab was associated with an increased risk of arterial thromboembolic events, especially in patients 65 years of age and older (8.5% vs. 2.9%, P<0.01). Because platelets play a crucial role in arterial thrombosis, we hypothesized that bevacizumab has direct platelet-stimulating activity. In a washed platelet system, bevacizumab alone had no effect on platelet aggregation. However, when combined with heparin (0.3 U/ml) and recombinant human VEGF (rhVEGF165 — a homodimeric protein with heparin binding sites) in a 1:2 molar ratio of antibody to antigen to allow optimal formation of immune complexes (ICs) in solution, bevacizumab potently induced platelet aggregation (up to 80–100%; n=5). Bevacizumab-induced platelet aggregation (BIPA) was functionally dependent on Fc domain binding to the platelet low-affinity IgG receptor, FcγRIIA, as demonstrated by an inhibitory monoclonal CD32 antibody (IV.3). BIPA was potentiated in platelets pre-sensitized with low concentrations of ADP or epinephrine. In contrast, BIPA was virtually absent at excess concentrations of heparin (100 U/ml), suggesting that translocation of ICs to the platelet surface via available heparin binding sites (on platelets) was crucial for this platelet response. Unfractionated heparin and the low-molecular-weight heparin, enoxaparin, were equally effective in promoting BIPA. In a manner similar to heparin-PF4 antibodies from patients with heparin-induced thrombocytopenia (HIT), bevacizumab-rhVEGF165-heparin ICs induced significant FcγRIIA-dependent dense granule release (>80%) in a 14C-serotonin release assay (SRA). While strong platelet responses were observed in both SRA and aggregometry, in which platelets were subjected to constant movement and low shear forces, respectively, bevacizumab-rhVEGF165-heparin ICs had only negligible effects on platelet CD62P expression under static conditions, indicating a critical role for platelet-platelet contacts in bevacizumab-mediated FcγRIIA signaling. In summary, our results suggest bevacizumab can induce strong FcγRIIA-dependent platelet activation in vitro when complexed with rhVEGF165 and heparin in an optimal stoichiometry. Due to their analogy to the pathomechanism of HIT, an acquired IgG-mediated disorder potentially associated with deleterious thrombosis, these findings may have direct clinical implications for older cancer patients with cardiovascular comorbidity, especially considering that many patients receive low doses of heparin for thromboprophylaxis and that elevated serum VEGF levels have been demonstrated in various types of malignancy, including CRC and NSCLC.

Platelet Activation by a Humanized Monoclonal CD40 Ligand (CD40L) Antibody.

Monoclonal antibodies are increasingly used as therapeutics, and some (anti-VEGF, anti-CD40L) have produced unexpected thrombotic side effects. For example, clinical trials with the humanized monoclonal CD40L antibody, hu5c8, in patients with systemic lupus erythematosus (SLE, a chronic inflammatory autoimmune disease) were prematurely terminated due to thromboembolic events. In SLE, elevated soluble CD40L (sCD40L) antigen levels may indicate in vivo platelet activation, putting patients at increased risk for thrombosis. We therefore hypothesized that anti-CD40L immunotherapy may represent an additional prothrombotic hit, leading to clinical manifestation of thromboembolic disease in patients with SLE. In this report, we provide experimental evidence that hu5c8, when combined with homotrimeric recombinant human sCD40L (rhsCD40L) in 1:3 molar ratio to allow formation of higher-order immune complexes (IC), activates platelets in an FcγRIIA-dependent manner. In a washed platelet system, IC formed of rhsCD40L (5 μg/mL) with hu5c8 (15 μg/mL) strongly induced platelet aggregation, while IC of rhsCD40L with hu5c8-Fab or hu5c8-F(ab')2 did not. Similarly, IC of rhsCD40L with hu5c8-agly, an aglycosylated hu5c8 variant with reduced Fc domain binding to the low affinity platelet IgG receptor, FcγRIIA (CD32), failed to induce aggregation. FcγRIIA dependency was further demonstrated with the monoclonal CD32 antibody, IV.3. These results suggest that hu5c8-mediated platelet activation requires both antigen (CD40L) and the IgG Fc domain. Flow cytometric analysis of P-selectin (CD62P) confirmed that IC of rhsCD40L with hu5c8, but not with Fab, F(ab')2, or hu5c8-agly, strongly activated platelets via FcγRIIA. When washed platelets were first sensitized with ADP, resulting in surface exposure of endogenous CD40L and sub-maximal aggregation of 5–25%, hu5c8 (10 μg/mL) induced strong platelet aggregation of up to 80–100% in the absence of exogenous rhsCD40L, indicating that physiologic levels of CD40L were sufficient for hu5c8-mediated platelet activation. ADP-sensitized platelets did not respond to equimolar concentrations of Fab, F(ab')2, or hu5c8-agly. In the serotonin release assay (SRA), which is used for the detection of FcγRIIA-activating heparin-platelet factor 4 (PF4) autoantibodies in patients with heparin-induced thrombocytopenia (HIT), the experimental procedure generates close platelet-platelet contacts and causes release of α-granule proteins such as CD62P, PF4, and CD40L. In the absence of rhsCD40L, hu5c8, but not Fab, F(ab')2, or hu5c8-agly, induced significant dense granule release, suggesting that exogenous rhsCD40L was not an absolute requirement for hu5c8-mediated platelet activation. In summary, our results show that the humanized monoclonal CD40L antibody, hu5c8, can strongly activate platelets in an Fc domain-dependent manner and that physiologically released CD40L is sufficient for this activity. This mechanism of activation is similar to that of HIT, in which homotetrameric PF4 released from prestimulated platelets provides the molecular basis for clustering of, and signaling through, FcγRIIA, resulting in a vicious cycle of platelet activation. Because thrombosis is a serious complication of HIT, it is reasonable to speculate that hu5c8-mediated platelet activation contributed to the pathophysiology of thromboembolic events in SLE patients receiving anti-CD40L immunotherapy.

Metabolism and Cytotoxicity of Menadione and Its Metabolite in Rat Platelets

Previous studies suggest that menadione is cytotoxic to rat platelets by oxidative stress. In order to elucidate the mechanism of this toxicity, metabolism of menadione and the cytotoxicity of a metabolite, menadione-glutathione conjugate (MEN-SG), were investigated in platelet rich plasma and washed platelet (WP) systems. When menadione was incubated in platelets, the primary metabolite was MEN-SG, which was excreted into the incubation medium. Incubation of subcellular fractions of platelets with synthetic MEN-SG led to increases in oxygen consumption that were similar to the parent compound, menadione. However, unlike menadione, exposure of MEN-SG to intact platelets in WP system neither resulted in increased oxygen consumption nor induced cell lysis as measured by lactate dehydrogenase leakage. In contrast to menadione, levels of MEN-SG in the incubation medium were unaffected by the presence of platelets, suggesting that MEN-SG was not consumed (or taken up) by platelets. These results indicate that even though MEN-SG was able to induce oxidative stress within platelets as potently as menadione itself, the MEN-SG formation from menadione in platelets appeared not to contribute to menadione's cytotoxicity. This lack of MEN-SG toxicity was likely due to its rapid excretion outside the cells.

Effects of nitric oxide/EDRF on platelet surface glycoproteins.

We examined the effects of nitric oxide (NO)/endothelium-derived relaxing factor (EDRF) on platelet surface glycoproteins (GP). As determined by flow cytometry, in both a washed platelet system and platelet-rich plasma, the EDRF congener (S-nitroso-N-acetylcysteine) markedly inhibited both the thrombin-induced and the (stable thromboxane A2 analogue) U-46619-induced upregulation of P-selectin (alpha-granule protein), CD63 (lysosomal protein), and the GPIIb-IIIa complex (fibrinogen receptor) but minimally inhibited downregulation of the GPIb-IX complex (von Willebrand factor receptor). The inhibitory effects of EDRF were markedly reduced in whole blood or by the addition of washed erythrocytes. Platelets in whole blood were still responsive to guanosine 3',5'-cyclic monophosphate (cGMP), as shown by complete inhibition of P-selectin upregulation by the stable analogue N6,2'-O dibutyryl cGMP. These data suggests that 1) cGMP negatively regulates the platelet surface expression of P-selectin, CD63, and the GPIIb-IIIa complex but not the platelet surface expression of the GPIb-IX complex and 2) hemoglobin within erythrocytes inhibits the effects of EDRF/NO on platelet surface glycoproteins.

The Activation-Induced Decrease in the Platelet Surface Expression of the Glycoprotein Ib-IX Complex Is Reversible

Thrombin decreases the platelet surface expression of the glycoprotein (GP) Ib-IX complex. To determine whether this effect is reversible, flow cytometric studies were performed with GPIb-IX-specific monoclonal antibodies. In both whole blood and washed platelet systems, incubation of platelets with thrombin or a combination of adenosine diphosphate and epinephrine resulted in a maximal decrease of the platelet surface expression of GPIb-IX within 5 minutes, after which there was a time-dependent return of the platelet surface GPIb-IX complex, which was maximal by 60 minutes. Exposure of the same platelets to additional exogenous thrombin resulted in a second decrease in platelet surface GPIb-IX, followed by a second reconstitution of platelet surface GPIb-IX. Throughout these experiments there was no measurable release from the platelets of glycocalicin (a proteolytic fragment of GPIb). Experiments in which platelets were preincubated with a biotinylated GPIb-specific MoAb showed that the GPIb molecules that returned to the platelet surface were the same molecules that had been translocated to the intraplatelet pool. The GPIb molecules that returned to the platelet surface were functionally competent to bind von Willebrand factor, as determined by ristocetin-induced platelet agglutination and ristocetin-induced binding of exogenous von Willebrand factor. Inhibitors of protein kinase C and myosin light-chain kinase enhanced the reexpression of platelet surface GPIb. In summary, the activation-induced decrease in the platelet surface expression of the GPIb-IX complex is reversible. Inactivation of protein kinase C and myosin light-chain kinase are important mechanisms in the reexpression of the platelet surface GPIb-IX complex.

The activation-induced decrease in the platelet surface expression of the glycoprotein Ib-IX complex is reversible

Thrombin decreases the platelet surface expression of the glycoprotein (GP) Ib-IX complex. To determine whether this effect is reversible, flow cytometric studies were performed with GPIb-IX-specific monoclonal antibodies. In both whole blood and washed platelet systems, incubation of platelets with thrombin or a combination of adenosine diphosphate and epinephrine resulted in a maximal decrease of the platelet surface expression of GPIb-IX within 5 minutes, after which there was a time- dependent return of the platelet surface GPIb-IX complex, which was maximal by 60 minutes. Exposure of the same platelets to additional exogenous thrombin resulted in a second decrease in platelet surface GPIb-IX, followed by a second reconstitution of platelet surface GPIb- IX. Throughout these experiments there was no measurable release from the platelets of glycocalicin (a proteolytic fragment of GPIb). Experiments in which platelets were preincubated with a biotinylated GPIb-specific MoAb showed that the GPIb molecules that returned to the platelet surface were the same molecules that had been translocated to the intraplatelet pool. The GPIb molecules that returned to the platelet surface were functionally competent to bind von Willebrand factor, as determined by ristocetin-induced platelet agglutination and ristocetin-induced binding of exogenous von Willebrand factor. Inhibitors of protein kinase C and myosin light-chain kinase enhanced the reexpression of platelet surface GPIb. In summary, the activation- induced decrease in the platelet surface expression of the GPIb-IX complex is reversible. Inactivation of protein kinase C and myosin light-chain kinase are important mechanisms in the reexpression of the platelet surface GPIb-IX complex.

Neonatal Platelets Are Less Reactive than Adult Platelets to Physiological Agonists in Whole Blood

Previous studies have reported that the platelets of healthy term neonates have either diminished or normal reactivity compared to the platelets of adults. To circumvent the methodologic problems of previous studies, we used a whole blood flow cytometric method to study neonatal platelet reactivity to thrombin, a combination of ADP and epinephrine, and U46619 (a stable thromboxane A2 analogue). Inclusion in the assay of the peptide GPRP (an inhibitor of fibrin polymerization) enabled us to study platelet reactivity to human alpha-thrombin in whole blood. Umbilical cord blood and day 1 peripheral blood were collected from 30 healthy term neonates and compared to peripheral blood from 20 normal adults. In whole blood samples without added agonist, there were no significant differences between neonates and adults in the platelet binding of monoclonal antibodies 6D1 (GPIb-specific) or 7E3 (GPIIb-IIIa complex-specific). As determined by S12 (a P-selectin-specific monoclonal antibody), neither neonates nor adults had circulating degranulated platelets. However, in both cord and peripheral whole blood samples, neonatal platelets were significantly less reactive than adult platelets to thrombin, ADP/epinephrine, and U46619, as determined by the extent of increase in the platelet surface expression of P-selectin and the GPIIb-IIIa complex, and the extent of decrease in the platelet surface expression of the GPIb-IX complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Heterogeneity of thromboxane A2 (TP‐) receptors: evidence from antagonist but not agonist potency measurements

1. Thromboxane A2 (TP-) receptors in human, rat and rabbit platelets and in smooth muscle of guinea-pig trachea, rat aorta and rabbit aorta have been characterized by measurement of the potencies of agonists and antagonists having considerable variations in chemical structure. 2. On each washed platelet system, eight prostanoids induced maximal irreversible aggregation (full agonists) and the potency ranking was EP 171 > STA2 > 9,11-azo PGH2 > 9,11-endoxy-10a-homo PGH2 > U-46619 (standard) > PGH2 = 16-p-fluorophenoxy-omega-tetranor PGF2 alpha > 16,16-dimethyl PGF2 alpha. Correlations between the three platelet preparations for both absolute and relative potencies were good. On human platelets, STA2, at concentrations above that required for maximum aggregation, exerted an inhibitory effect which was independent of its interaction with the TP-receptor. 3. Five prostanoids, EP 109, EP 167, EP 204, PTA2 and 16,20-methano PTA2, exhibited partial agonist activity on the platelet and smooth muscle preparations. There was good agreement between absolute potencies on the six preparations; on platelets potency was assessed from shape change measurements, since aggregation, when present, always showed a very shallow concentration-response relationship. The magnitude of the maximum response induced by each compound decreased in the order listed above, to the extent that 16,20-methano PTA2 could be treated as a pure antagonist. 4. With U-46619 as agonist, the pA2 values of seven antagonists were found to be very similar on human and rat platelets. The potency ranking was EP 169 > AH 23848 > EP 092 > ONO 11120 > EP 115 = 16,20-methano PTA2 > BM 13177. There was a similar trend on rabbit platelets but pA2 values were 1.0-1.5 log units smaller; the exception was BM 13177 which had similar affinities. The antagonism produced by EP 169 and AH 23848 was surmountable on rabbit platelets but not on human and rat platelets. 5. None of the antagonists was highly potent on the rabbit aorta (pA2 values < 7.5 by Schild analysis). Affinities on the guinea-pig trachea and the rat aorta were higher and in the same range as those obtained for human and rat platelets. However the correlations of pA2 values between any pair of smooth muscle preparations and between any pair of platelet/smooth muscle preparations were either weak or not significant(P > 0.05). 6. The excellent agreement for both full and partial agonist potencies between the six preparations provides no evidence for TP-receptor subtypes and further suggests that the agonist recognition sites of the TP-receptors could be very similar, if not identical, in nature. In contrast, the different antagonist affinities found in this and other published studies indicate heterogeneity of TP-receptors. However, classification into TP,-, TP2-receptors, etc. on the basis of the limited antagonist data available does not appear appropriate at this time.

Downregulation of the Platelet Surface Glycoprotein Ib-IX Complex in Whole Blood Stimulated by Thrombin, Adenosine Diphosphate, or an In Vivo Wound

In washed platelet systems, thrombin has been demonstrated to downregulate the platelet surface expression of glycoprotein (GP) Ib and GPIX. In the present study, we addressed the question as to whether, in the more physiologic milieu of whole blood, downregulation of platelet surface GPIb and GPIX can be induced by thrombin, adenosine diphosphate (ADP), and/or by an in vivo wound. Thrombin-induced downregulation of GPIb and GPIX on the surface of individual platelets in whole blood was demonstrated by the use of flow cytometry, a panel of monoclonal antibodies (MoAbs) and, to inhibit fibrin polymerization, the peptide glycyl-L-prolyl-L-arginyl-L-proline. Platelets were identified in whole blood by a GPIV-specific MoAb and exclusion of monocytes by light scattering properties. Flow cytometric analysis of whole blood emerging from a standardized bleeding-time wound established that downregulation of platelet surface GPIb and GPIX can occur in vivo. A GPIb-IX complex-specific antibody indicated that the GPIb and GPIX remaining on the surface of platelets activated in vivo or in vitro were fully complexed. Simultaneous analysis of individual platelets by two fluorophores demonstrated that thrombin-induced platelet surface exposure of GMP-140 (degranulation) was nearly complete at the time that downregulation of platelet surface GPIb-IX was initiated. However, degranulation was not a prerequisite because ADP downregulated platelet surface GPIb-IX without exposing GMP-140 on the platelet surface. Inhibitory effects of cytochalasins demonstrated that the activation-induced downregulation of both GPIX and GPIb are dependent on actin polymerization. In summary, downregulation of the platelet surface GPIb-IX complex occurs in whole blood stimulated by thrombin, ADP, or an in vivo wound, and is independent of alpha granule secretion.

Downregulation of the platelet surface glycoprotein Ib-IX complex in whole blood stimulated by thrombin, adenosine diphosphate, or an in vivo wound [see comments

In washed platelet systems, thrombin has been demonstrated to downregulate the platelet surface expression of glycoprotein (GP) Ib and GPIX. In the present study, we addressed the question as to whether, in the more physiologic milieu of whole blood, downregulation of platelet surface GPIb and GPIX can be induced by thrombin, adenosine diphosphate (ADP), and/or by an in vivo wound. Thrombin-induced downregulation of GPIb and GPIX on the surface of individual platelets in whole blood was demonstrated by the use of flow cytometry, a panel of monoclonal antibodies (MoAbs) and, to inhibit fibrin polymerization, the peptide glycyl-L-prolyl-L-arginyl-L-proline. Platelets were identified in whole blood by a GPIV-specific MoAb and exclusion of monocytes by light scattering properties. Flow cytometric analysis of whole blood emerging from a standardized bleeding-time wound established that downregulation of platelet surface GPIb and GPIX can occur in vivo. A GPIb-IX complex-specific antibody indicated that the GPIb and GPIX remaining on the surface of platelets activated in vivo or in vitro were fully complexed. Simultaneous analysis of individual platelets by two fluorophores demonstrated that thrombin-induced platelet surface exposure of GMP-140 (degranulation) was nearly complete at the time that downregulation of platelet surface GPIb-IX was initiated. However, degranulation was not a prerequisite because ADP downregulated platelet surface GPIb-IX without exposing GMP-140 on the platelet surface. Inhibitory effects of cytochalasins demonstrated that the activation-induced downregulation of both GPIX and GPIb are dependent on actin polymerization. In summary, downregulation of the platelet surface GPIb-IX complex occurs in whole blood stimulated by thrombin, ADP, or an in vivo wound, and is independent of alpha granule secretion.

The role of fibronectin in platelet aggregation

A monoclonal antibody (anti-Fn2) was prepared which was reactive with both plasma fibronectin and fibronectin located within the platelet alpha granule. Immunoblotting analysis, on thermolysin digestion fragments of fibronectin, identified two immunoreactive fragments of Mr 145 kDa and 155 kDa which are known to contain a cell and DNA binding region. Anti-Fn2 was found to inhibit binding of fibronectin to platelets and DNA. Functional platelet studies, measuring platelet aggregation and 14C-serotonin release in washed platelet systems, demonstrated the ability of anti-Fn2 to totally inhibit low dose thrombin and low-dose collagen induced platelet aggregation and serotonin release. Anti-Fn2 partially inhibited platelet aggregation induced by ADP (10 microM) and arachidonic acid, but had no effect on platelet aggregation induced by high-dose thrombin or by the calcium ionophore A23187. These studies indicate that fibronectin participates in platelet aggregation and release induced by a range of agonists and suggest that it has a more important involvement in platelet function than previously described.

Platelet Recovery From Aspirin Inhibition In Vivo; Differing Patterns Under Various Assay Conditions

The linear return of platelet malondialdhyde (MDA) production after aspirin ingestion as described by Stuart et al.4 has been widely accepted as a nonisotopic method for the determination of platelet survival. However, using assays for platelet prostaglandin production and cyclooxygenase activity, a number of authors2,9–11 have now reported a 24 to 48-hr delay in recovery after aspirin. This delay has been interpreted as evidence that aspirin inhibits megakaryocyte as well as platelet cyclooxygenase. The discrepancy in these findings has not been explained. We have measured the return of production of MDA and thromboxane B2 (TXB2) by platelets of normal volunteers after a single dose of aspirin using a variety of assay conditions. We find striking differences in the pattern of recovery depending upon the presence of protein in the medium in which the platelets are stimulated. Production of both MDA and TXB2 returns in a linear fashion with time when platelets are washed, resuspended in buffer and stimulated with either NEM or thrombin. A 48-hr lag in recovery is seen when platelets are stimulated in whole blood or platelet-rich plasma, or when washed platelets are resuspended in either platelet-poor plasma or in buffer containing 4 g % albumin. This lag is present regardless of stimulus or metabolite measured. It does not appear to be an artifact resulting from binding of the arachidonic acid or its metabolic products to the protein in the medium, as we are able to detect TXB2 production by even small numbers of aspirin-free platelets in whole blood mixtures. Therefore, if megakaryocyte cyclooxygenase is inhibited by aspirin, this effect is obscured when the platelets are suspended in protein-free medium. We have also studied a number of patients with short platelet survivals as judged by Cr51 labeling. Survivals using the aspirin method and washed platelets in these patients are consistently longer than those determined simultaneously by Cr51. This leads us to postulate that there is a lag effect even in the washed platelet system, and that this effect is more evident in states of increased platelet turnover. This complex effect must be better understood before aspirin platelet survivals can be accurately interpreted.

Release of VIII R:Ag and VIII R:WF during thrombin and collagen induced aggregation

Platelet VIII R:Ag and VIII R:WF release was measured during platelet aggregation using collagen and thrombin. In a washed platelet system it was shown that normal platelets released one fourth to one third of the VIII R:Ag and VIII R:WF measured in the platelet extract after freezing and thawing. Platelets from patients with storage pool disease or patients treated with aspirin did not release VIII R:Ag or VIII R:WF using collagen and only a small amount with thrombin. VIII R:Ag and VIII R:WF were not released from platelets of patients with von Willebrand disease even from platelets of those patients who showed normal amount of platelet VIII R:Ag.

Reversible and irreversible inhibition of platelet cyclooxygenase and serotonin release by nonsteroidal antiinflammatory drugs

Indomethacin, fenoprofen, naproxen, acetylsalicylic acid, phenylbutazone and sulfinpyrazone were studied as inhibitors of serotonin release and of cyclooxygenase activity in human platelets. Inhibition by indomethacin, acetylsalicylic acid and naproxen, but not the other drugs, was time-dependent and irreversible. When release of serotonin- 14C was induced by dilute collagen all drugs produced concentration-dependent inhibition which became virtually complete at higher drug concentrations. The rank order of potency of the inhibitors was similar to that observed for cyclooxygenase activity of washed platelets for most drugs. These observations indicate the importance of arachidonate metabolism as the principal pathway mediating the action of dilute collagen. Acetylsalicylic acid which is relatively loosely bound to albumin inhibited release in plasma and enzyme activity in washed platelets with approximately equal potency. The other drugs are more avidly bound to albumin, and concentrations required to inhibit release were 10 to 50 times greater than the concentrations which produced a similar degree of inhibition of cyclooxygenase activity in the washed platelet system. The potency of the drugs was compared with their potency as inhibitors of cyclooxygenase activity of gastric mucosa. The effect of fenoprofen and of sulfinpyrazone was considerably stronger in the platelet system, while the other drugs exhibited activity which was comparable on the 2 tissues.

Von Willebrand-Jürgens syndrome with a variant of factor VIII-associated antigen

A family is described in which 5 out of 8 children had a marked bleeding disorder. The children showed prolonged bleeding times, abnormal platelet retention upon passage of blood through a glass bead column, the Willebrand factor activity as measured by ristocetin in a washed platelet system was low. Factor VIII/von Willebrand factor protein levels were normal even so the factor VIII-procoagulant activity. Even the parents and one child without any bleeding tendency and normal bleeding times had a reduced Willebrand factor activity. In all these patients evidence of an abnormal protein was observed on crossed antigen-antibody electrophoresis indicating a qualitative defect of the factor VIII/von Willebrand factor protein.

Studies of the Mechanisms of the Human Platelet Release Reaction Induced by Immunologic Stimuli

Abstract Human platelets in the presence of plasma show a biphasic response to immune complexes (IC) formed from either antigen and antibody or from aggregated immunoglobulin. Rapid release of serotonin within the 1st min is unaffected by cobra venom factor (CVF), EDTA, and agents which prevent the aggregating action of ADP, but is inhibited by complement (C) components. This release appears to be the direct interaction of platelets and IC analogous to release by washed platelets which is inhibited by C1. Aggregation follows, accompanied by a second release which requires ADP and is inhibited by CVF and EDTA. It is dependent on C, activated most probably via the classical pathway. Zymosan (Z) causes aggregation and release with the characteristics of the second phase of IC-induced release. This reaction, which occurs after an 8 to 10 minute lag phase, has an absolute requirement for C components which may be activated by the C3 shunt. Z binds the necessary components such that the resulting complex (Z-X) induces aggregation and release in a washed platelet system. Divalent metal ions and ADP are necessary for platelet response to Z-X.

Thrombin-induced increase in intracellular cyclic 3',5'-adenosine monophosphate in human platelets.

The present data disagree with earlier suggestions that thrombin's effect on platelets is to cause a decrease in intracellular cyclic 3',5'-adenosine monophosphate. Washed human platelets or platelet-rich plasma were incubated at 37 degrees C with human thrombin. After centrifugation, the supernates were assayed for nucleotides and calcium released. The platelet pellets, and in some experiments the supernates as well, were assayed by radioimmunoassay for intracellular cyclic AMP. In the washed platelet system, increasing doses of thrombin to 0.5 U/cc induced increasing release of nucleotides and calcium. This was accompanied by an average twofold increase in intracellular cyclic AMP levels. Prostaglandin E(1), which inhibited 30-50% of release, induced a four- to fivefold increase in cyclic AMP levels that was additive to the cyclic AMP-stimulatory effect of thrombin. Theophylline, which inhibited only 20-40% of nucleotide release, was synergistic with thrombin in the intracellular accumulation of cyclic AMP. The time-course of cyclic AMP accumulation in response to thrombin was slower than thrombin-induced nucleotide release. Similar findings were made in the platelet-rich plasma system where thrombin stimulation of nucleotide release also resulted in a marked accumulation of intracellular cyclic AMP. Thrombin did not appear to stimulate the release of intracellular cyclic AMP. The mechanism underlying these observations was not apparent. The thrombin had no measurable inhibitory effect on platelet phosphodiesterase activity in either intact washed cells or the platelet homogenate supernates. Furthermore, thrombin inhibited, rather than stimulated, platelet adenyl cyclase activity in both intact washed cells and washed platelet particulate fractions. Of note, however, was the finding that thrombin did not completely inhibit the adenyl cyclase activity of prostaglandin-stimulated cells. Further work is needed to clarify the significance of this observation.Nonetheless, the accumulation of intracellular cyclic AMP in response to thrombin observed in the present study suggests that the antagonistic actions of various agents on the platelet release reaction, thought to underlie platelet function, may depend upon a mechanism more intricate than a straightforward mediation through directly opposite effects on platelet cyclic AMP.